Polyhydroxyalkanoate, method for production thereof and microorganisms for use in the same

ABSTRACT

A polyhydroxyalkanoate having a monomer unit composition represented by General Formula (1): 
     
       
         A m B (1−m)   (1) 
       
     
      wherein 
     A is represented by General Formula (2), B is at least one selected from the group consisting of monomer units represented by General Formula (3) or (4), and m has a value of 0.01 or larger and smaller than 1:                    
      wherein 
     n has a value of 0 to 10, k has a value of 3 or 5, and 
     R is at least one group selected from the group consisting of groups represented by General Formulae (5) to (7):                    
      in Formula (5) 
     R1 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, —CN, —NO 2 , —CF 3 , —C 2 F 5  and —C 3 F 7 ; and q is an integer selected from 1 to 8; 
      in Formula (6) 
     R2 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, —CN, —NO 2 , —CF 3 , —C 2 F 5  and —C 3 F 7 ; and r is an integer selected from 1 to 8; 
      in Formula (7) 
     R3 is a group selected from the group consisting of a hydrogen atom (H), halogen atoms, —CN, —NO 2 , —CF 3 , —C 2 F 5  and —C 3 F 7 ; and s is an integer selected from 1 to 8. 
     The efficient production methods are also provided.

This application is a division of Application No. 09/748,205, filed Dec.27, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel polyhydroxyalkanoate (PHA), amethod for production of such PHA and microorganisms for use in thesame.

2. Related Background Art

Synthetic polymers derived from petroleum have been used as plasticsetc. for a long time. Recently, the treatment of the used plastics hasbecome one of serious social problems. These synthetic polymers haveadvantages of hard-to-decompose have been used in the place of metal orglass materials. On mass consumption and mass disposal, however, thisfeature of hard-to-decompose makes them accumulated in waste-disposalfacilities, or when they are burned, it causes increased carbon dioxideexhaust, and harmful substances such as dioxin and endocrine-disruptorsmay be generated to cause environmental pollution. On the other hand,polyhydroxyalkanoates (PHAs) produced by microorganisms (hereinafterreferred to as “microbial polyester”) represented by poly-3-hydroxybutyric acid (PHB) can be used as the conventional plastics to makevarious kinds of products with melting processes etd., and can bedecomposed by organisms unlike oil-derived synthetic polymers.Therefore, the microbial polyester is bio-decomposed and thusincorporated in the natural material cycle when discarded, and would notremain in the natural environment to cause pollution unlike manyconventional synthetic polymer compounds. Furthermore, since themicrobial polyesters do not require incineration processes, they arealso effective in terms of prevention of air pollution and globalwarming. Thus, they can be used as a plastic enabling environmentalintegrity. In addition, the application of the microbial polyesters tomedical soft members is under consideration (Japanese Patent ApplicationLaid-Open No. 5-159, Japanese Patent Application Laid-Open No. 6-169980,Japanese Patent Application Laid-Open No. 6-169988, Japanese PatentApplication Laid-Open No. 6-225921 and the like).

Heretofore, various bacteria have been reported to produce andaccumulate PHB or copolymers of other hydroxyalkanoic acids in the cells(“Biodegradable Plastics Handbook”, edited by Biodegradable PlasticsSociety, issued by NTS Co. Ltd., P178-197, (1995)). It is known thatsuch microbial PHAs may have a variety of compositions and structuresdepending on types of the producing microorganisms, the composition ofculture media, culture conditions and the like, and up to now, studiesregarding the control of these compositions and structures have beencarried out to improve the properties of PHA.

For example, Alcaligenes eutropus H16 (ATCC No. 17699) and its mutantstrains reportedly produce copolymers of 3-hydroxy butyric acid (3HB)and 3-hydroxy valeric acid (3HV) at a variety of composition ratiosaccording to the carbon source in culture (Japanese Patent PublicationNo. 6-15604, Japanese Patent Publication No. 7-14352, Japanese PatentPublication No. 8-19227 and the like).

Japanese Patent Application Laid-Open No. 5-74492 discloses a method inwhich the copolymer of 3HB and 3HV is produced by bringingMethylobacterium sp., Paracoccus sp., Alcalugenes sp. or Pseudomonas sp.into contact primary alcohol having 3 to 7 carbons.

Japanese Patent Application Laid-Open No. 5-93049 and Japanese PatentApplication Laid-Open No. 7-265065 disclose that two-componentcopolymers of 3HB and 3-hydroxy hexanoic acid (3HHx) are produced byculturing Aeromonas caviae using oleic acid or olive oil as a carbonsource.

Japanese Patent Application Laid-Open No. 9-191893 discloses thatComamonas acidovorans IFO 13852 produces polyester having 3HB and4-hydroxy butyric acid as monomer units in culture with gluconic acidand 1,4-butandiol as a carbon source.

Also, in recent years, active researches about PHA composed of3-hydroxyalkanoate (3HA) of medium-chain-length (abbreviated to mcl)having up to about 12 carbons. Synthetic routes can be classifiedbroadly into two types, and their specific examples will be shown in (1)and (2) below.

(1) Synthesis Using β-oxidation

Japanese Patent No. 2642937 discloses that PHA having monomer units of3-hydroxyalkanoate having 6 to 12 carbons is produced by providing as acarbon source aliphatic hydrocarbon to Pseudomonas oleovorans ATCC29347. Furthermore, it is reported in Appl. Environ. Microbiol, 58(2),746 (1992) that Pseudomonas resinovorans produces polyester having3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acidand 3-hydroxydecanoic acid at a ratio of 1:15:75:9 as monomer units,using octanoic acid as a single carbon source, and also producespolyester having 3-hydroxybutyric acid, 3-hydroxyhexanoic acid,3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (quantitative ratio of8:62:23:7) as units, using hexanoic acid as a single carbon source.Herein, it is assumed that 3HA monomer units having longer chain lengththan that of the starting fatty acid are made by way of fatty acidsynthetic route that will be described next in (2).

(2) Synthesis Using Fatty Acid Synthetic Route

It is reported in Int. J. Biol. Macromol., 16(3), 119 (1994) thatPseudomonas sp. 61-3 strain produces polyester made of 3-hydroxyalkanoicacids such as 3-hydroxybutyric acid, 3-hydroxyhexanoic acid,3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoicacid and 3-hydroxyalkenoic acids such as 3-hydroxy-5-cis-decenoic acid,3-hydroxy-5-cis-dodecenoic acid, using sodium gluconate as a singlecarbon source.

By the way, the biosynthesis of PHA is usually carried out by a PHAsynthase using as a substrate “D-3-hydroxyacyl-CoA” occurring as anintermediate of a variety of metabolic pathways in the cell.

Herein, “CoA” means a “coenzyme A”. And, as described in the prior artof the above (1), the biosynthesis of PHA is carried out with“D-3-hydroxyacyl-CoA” occurring in the “β oxidation cycle” being astarting substance in the case where fatty acids such as octanoic acidand nonanoic acid are used as carbon sources.

Reactions through which PHA is synthesized by way of the “β oxidationcycle” will be shown below.

On the other hand, as described in the prior art of the above (2), inthe case where the PHA is biosynthesized using saccharides such asglucose and the like, the biosynthesis is carried out with“D-3-hydroxyacyl-CoA” converted from “D-3-hydroxyacyl-ACP” occurring inthe “fatty acid synthesis pathway” being a starting substance. Herein,“ACP” means a “acyl carrier protein”.

By the way, as described previously, the PHA synthesized in both (1) and(2) described above is PHA constituted by monomer units having alkylgroups in side chains. However, if a wider range of application of themicrobial PHA like this, for example an application as a functionalpolymer is considered, it is expected that PHA having varioussubstituents (for example phenyl groups) introduced in the side chain issignificantly useful. With respect to the synthesis of such PHA, for thesynthesis using P oxidation, a report regarding PHA having the arylgroup and the like in the side chain can be found in, for example,Macromolecules, 24, p5256-5260 (1991). Specifically, it is reported thatPseudomonas oleovorans produces polyester having 3-hydroxy valeric acid,3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, 3-hydroxyundecanoicacid and 3-hydroxy-5-phenyl valeric acid (quantitative ratio of0.6:16.0:41.1:1.7:40.6) as units in the amount of 160 mg for 1 L ofculture solution (ratio in dry weight to the cell mass is 31.6%), using5-phenylvaleric acid and nonanoic acid (mole ratio of 2:1, totalconcentration of 10 mmol/L) as a medium, and also produces polyesterhaving 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoicacid and 3-hydroxy-5-phenyl valeric acid (quantitative ratio of7.3:64.5:3.9:24.3) as units in the amount of 200 mg for 1 L of culturesolution (ratio in dry weight to the cell mass is 39.2%), using 5-phenylvaleric acid and octanoic acid (mole ratio of 1:1, total concentrationof 10 mmol/L). It is conceivable that the PHA in this report isprincipally synthesized by way of the β oxidation pathway due to thatfact that nonanoic acid and octanoic acid are used.

As described above, in the microbial PHA, those with various kinds ofcompositions/structures are obtained by changing the type ofmicroorganisms for use in its production, culture medium compositions,culture conditions, but if considering the application of the microbialPHA as plastics, they could not be sufficient yet in terms ofproperties. In order to further expand the range of the microbial PHAutility, it is important that the improvement of its properties are morewidely considered, and for this purpose, the development and the searchof the PHA containing monomer units of further diverse structures, itsmanufacturing processes and microorganisms enabling desired PHA to beproduced efficiently are essential.

On the other hand, the PHA of a type having substituents introduced inthe side chain as described previously is selected in accordance withthe property for which the introduced substituent is desired, therebymaking it possible to expect its development as a “functional polymer”having very useful functions and properties resulting from the propertyand the like of the introduced substituent, and the development and thesearch of excellent PHA allowing such functionality and thebiodegradability to be compatible with each other, its manufacturingprocesses and microorganisms enabling desired PHA to be producedefficiently are also important challenges.

Another example of such PHA having substituents introduced in the sidechain includes PHA having the above described phenyl groups, and furtherphenoxy groups in the side chain.

For another example of phenyl group, it is reported in Macromolecules,29, 1762-1766 (1996) that Pseudomonas oleovorans produces PHA including3-hydroxy-5-(4-toryl)valeric acid as a monomer unit through the culturein a culture medium including 5-(4-toryl) valeric acid(5-(4-methylphenyl)valeric acid) as a substrate.

Furthermore, it is reported in Macromolecules, 32, 2889-2895 (1999) thatPseudomonas oleovorans produces PHA including3-hydroxy-5-(2,4-dinitrophenyl)valeric acid and3-hydroxy-5-(4-nitrophenyl)valeric acid as monomer units through theculture in a culture medium including 5-(2,4-dinitrophenyl)valeric acidand nonanoic acid as a substrate.

Also, for an example of the phenoxy group, it is reported in Macromol.Chem. Phys., 195, 1665-1672 (1994) that Pseudomonas oleovorans producesPHA including 3-hydroxy-5-phenoxy valeric acid and3-hydroxy-9-phenoxynonanoic acid as units from 11-phenoxyundecanoicacid.

Also, it is reported in Macromolecules, 29, 3432-3435 (1996) thatPseudomonas oleovorans is used to produce PHA including3-hydroxy-4-phenoxybutyric acid and 3-hydroxy-6-phenoxyhexanoic acid asunits from 6-phenoxyhexanoic acid, PHA including3-hydroxy-4-phenoxybutyric acid, 3-hydroxy-6-phenoxyhexanoic acid and3-hydroxy-8-phenoxyoctanoic acid as units from 8-phenoxyoctanoic acid,and PHA including 3-hydroxy-5-phenoxyvaleric acid and3-hydroxy-7-phenoxyheptanoic acid as units from 11-phenoxyundecanoicacid. Excerpts of yields of polymers from this report are shown in Table1.

Furthermore, in Can. J. Microbiol., 41, 32-43 (1995), PHA including3-hydroxy-p-cyanophenoxyhexanoic acid or3-hydroxy-p-nitrophenoxyhexanoic acid as a monomer unit is successfullyproduced with octanoic acid and p-cyanophenoxyhexanoic acid orp-nitrophenoxyhexanoic acid being a substrate, using Pseudomonasoleovorans ATCC 29347 and Pseudomonas putida KT 2442.

In Japanese Patent No. 2989175, a homopolymer constituted by3-hydroxy-5-(monofluorophenoxy)pentanoate(3H5(MFP)P) units or3-hydroxy-5-(difluorophenoxy)pentanoate(3H5(DFP)P) units and a copolymercontaining at least 3H5(MFP)P units or 3H5(DFP)P units; Pseudomonasputida for synthesizing these polymers; and a method of producing theaforesaid polymers using Pseudomonas species are described.

These productions are carried out through “two-stage culture” asdescribed below. Time of culture: First stage, 24 hours; Second stage,96 hours.

A substrate and a resulting polymer at each stage will be shown below.

(1) Resulting Polymer: 3H5 (MFP) P Homopolymer

Substrate at the first stage: Citric acid, Yeast extract

Substrate at the second stage: Monofluorophenoxyundecanoic acid

(2) Resulting Polymer: 3H5 (DFP) P Homopolymer

Substrate at the first stage: Citric acid, Yeast extract

Substrate at the second stage: Difluorophenoxyundecanoic acid

(3) Resulting Polymer: 3H5 (MFP) P Copolymer

Substrate at the first stage: Octanoic acid or Nonanoic acid, Yeastextract

Substrate at the second stage: Monofluorophenoxyundecanoic acid

(4) Resulting Polymer: 3H5 (DFP) P copolymer

Substrate at the first stage: Octanoic acid or Nonanoic acid, Yeastextract

Substrate at the second stage: Difluorophenoxyundecanoic acid

As its effect, a medium-chain-length fatty acid having substituents maybe materialized to synthesize a polymer having phenoxy groups with endsof the side chain replaced by one to two fluorine atoms, andstereoregularity and water repellency can be provided while maintaininga high melting point and good processability.

Also, PHA including cyclohexyl groups in monomer units is expected toshow polymeric properties different from those of PHA including normalaliphatic hydroxyalkanoic acid as a unit, and an example of productionusing Pseudomonas oleovorans has been reported (Macromolecules, 30,1611-1615 (1997)).

According to this report, when Pseudomonas oleovorans was cultured in aculture medium where nonanoic acid (hereinafter described as NA) andcyclohexylbutyric acid (hereinafter described as CHBA) or cyclohexylvaleric acid (hereinafter described as CHVA) coexisted, PHA includingunits containing cyclohexyl groups and units originating from nonanoicacid were obtained (each ratio unknown)

About the yields, it is reported that quantitative ratios of CHBA and NAare varied with substrate concentration total of 20 mmol/L and resultsas shown in Table 2 were obtained.

However, in this example, the yield of polymers per culture solution isnot sufficient, and the obtained PHA itself has aliphatichydroxyalkanoic acid coexist in its monomer unit.

In this way, in the case where PHA with a variety of substituentsintroduced in the side chain is produced, as seen in the reportedexample of Pseudomonas oleovorans described previously and the like, amethod is used in which alkanoate having a substituent to be introducedis used not only as a stock for the polymer but also as a carbon sourcefor growth.

However, for the method in which alkanoate having a substituent to beintroduced is used not only as a stock for the polymer but also as acarbon source for growth, the supply of an energy source based on theproduction of the acetyl-CoA by β oxidation from such alkanoate isexpected, and in this method, only a substrate having a certain degreeof chain length is capable of producing acetyl-CoA by β oxidation, thuslimiting alkanoate that can be used as a substrate of PHA, which is amajor problem. Also, generally, since substrates with the chain lengthdecreased by two methylene chains an after another are newly produced bythe β oxidation, and these are captured as monomer units of PHA, the PHAthat is synthesized is often a copolymer constituted by monomer unitsthat are different in the chain length by two methylene chains one afteranother. In the reported example described above, a copolymerconstituted by three types of monomer units, that is3-hydroxy-8-phenoxyoctanoic acid originating from 8-phenoxyoctanoic acidwhich is a substrate, 3-hydroxy-6-phenoxyhexanoic acid and3-hydroxy-4-phenoxybutyric acid which are by-products originating frommetabolites is produced. In this respect, if PHA constituted by singlemonomer units is to be obtained, it is quite difficult to use thismethod. Furthermore, for a method premised on the supply of an energysource based on the production of acetyl-CoA by the β oxidation, thegrowth of microorganisms is slow and the synthesis of PHA requires lotsof time, and the yield of the synthesized PHA is often low, which isalso a major problem.

For this reason, a method in which, in addition to the alkanoate havingsubstituents to be introduced, microorganisms are cultured in theculture medium in which fatty acids of medium-chain-length and the likesuch as octanoic acid and nonanoic acid as the carbon source for growth,followed by extracting PHA is considered to be effective and isgenerally used.

However, according to the study by the inventors, the PHA synthesized byway of the β oxidation pathway using fatty acids of medium-chain-lengthsuch as octanoic acid and nonanoic acid as the carbon source for growthhas poor purity, and 50% or more of the polymers are made of mcl-3HAmonomer units originating from the carbon source (for example,3-hydroxyoctanoic acid, 3-hydroxynonanoic acid and the like). Thesemcl-3HA units make polymers adhesive at room temperature when they aresole components, and if they coexist in the PHA of the present inventionin large quantity, the glass transition temperature (Tg) of the polymeris significantly lowered. Thus, to obtain hard polymers at roomtemperature, the coexistence of mcl-3HA monomer units is not desired.Also, it is known that such a hetero-side chain structure interferesintra-molecular or inter-molecular interaction originating from the sidechain structure, and has significant influence on crystallinity andorientation. For achieving the improvement of polymer properties and theaddition of functionality, the coexistence of these mcl-3HA monomerunits raises a major problem. Means for solving this problem includesproviding a refinement process to separate/remove “undesired” monomerunits such as mcl-3HA monomer units originating from the carbon sourcefor growth, in order to acquire PHA constituted by monomer units havingonly specified substituents. However, the problem is that operations arecomplicated and a significant decrease in the yield can not be avoided.A more serious problem is that if desired monomer units and undesiredmonomer units form a copolymer, it is quite difficult to remove onlyundesired monomer units. Particularly, in the case where the purpose isto synthesize PHA including monomer units having groups obtained fromunsaturated hydrocarbons, ester groups, aryl groups, cyan groups, nitrogroups, groups obtained from halogenated hydrocarbons, groups havingepoxide and the like introduced therein as side chain structures, themcl-3HA monomer unit often forms a copolymer with a desired monomerunit, and it is extremely difficult to remove the mcl-3HA monomer unitafter PHA is synthesized.

SUMMARY OF THE INVENTION

The present invention solves the above described problems, and providesPHA including monomer units of diverse structures having substituents inthe side chain, which is useful as device materials, medical materialsand the like, and a method of producing such PHA using microorganisms,and particularly a production method in which the coexistence ofundesired monomer units is reduced, desired PHA can be obtained in highpurity and also in high yields. The present invention is also intendedto provide strains enabling such PHA to be synthesized in high purityand with efficiency.

According to one aspect of the present invention, there is provided apolyhydroxyalkanoate having a monomer unit composition represented by ageneral formula (1):

A_(m)B_((1−m))  (1)

 wherein

A is represented by General Formula (2), B is at least one selected fromthe group consisting of monomer units represented by General Formula (3)or (4), and m has a value of 0.01 or larger and smaller than 1:

 wherein

n has a value of 0 to 10, k has a value of 3 or 5, and

R is at least one group selected from the group consisting of groupsrepresented by General Formulae (5) to (7):

 in Formula (5)

R1 is a group selected from the group consisting of a hydrogen atom (H),halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; and q is an integerselected from 1 to 8;

 in Formula (6)

R2 is a group selected from the group consisting of a hydrogen atom (H),halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; and r is an integerselected from 1 to 8;

 in Formula (7)

R3 is a group selected from the group consisting of a hydrogen atom (H),halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; and s is an integerselected from 1 to 8;

provided that following R are excluded from the choice:

when selecting one type of group as R in the general formula (2):

groups of Formula (5) in which R1 is H and q=2, R1 is H and q=3, and R1is —NO₂ and q=2;

groups of Formula (6) in which R2 is a halogen atom and r=2, providedthat two components are selected as B from General Formula (3) or (4),R2 is —CN and r=3, and R2 is —NO₂ and r=3; and the groups of Formula (7)in which R3 is H and s=1, and R3 is H and s=2; and

when selecting two types of groups as R in General Formula (2), thecombinations of two types of groups of Formula (6) in which R2 is ahalogen atom and r=2, and R2 is a halogen atom and r=4, provided thatone component is selected as B from General Formula (3) or (4).

According to one aspect of the present invention, there is provided5-(4-trifluoromethylphenyl)valeric acid of Formula (21).

According to another aspect of the present invention, there is provideda method of producing a polyhydroxyalkanoate, comprising a step ofculturing a microorganism capable of synthesizing a polyhydroxyalkanoateof which monomer unit is represented by Formula (1) from an alkanoate ina medium containing the alkanoate:

A_(m)B_((1−m))  (1)

 wherein

A is represented by General Formula (2), B is at least one selected fromthe group consisting of monomer units represented by General Formula (3)or (4), and m is 0.01 or larger and smaller than 1,

 wherein

n is an integer selected from 0 to 10, k is 3 or 5, and

R is at least one group selected from the group consisting of the groupsrepresented by General Formulae (5) to (7):

 in Formula (5)

R1 is a group selected from the group consisting of a hydrogen atom (H),halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; and q is an integerselected from 1 to 8;

 in Formula (6)

R2 is a group selected from the group consisting of a hydrogen atom (H),halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; and r is an integerselected from 1 to 8;

 in Formula (7)

R3 is a group selected from the group consisting of a hydrogen atom (H),halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; and s is an integerselected from 1 to 8;

provided that following R are excluded from the choice:

when selecting one type of group as R in General Formula (2):

groups of Formula (5) in which R1 is H and q=2, R1 is H and q=3, and R1is —NO₂ and q 2; the groups of Formula (6) in which R2 is a halogen atomand r=2, R2 is —CN and r=3, and R2 is —NO₂ and r=3; and

groups of Formula (7) in which R3 is H and s=1, and R3 is H and s=2; and

when selecting two types of groups as R in General Formula (2),

groups of Formula (6) in which R2 is a halogen atom and r=2.

According to a further aspect of the present invention, there isprovided a process of producing polyhydroxyalkanoate, comprising a stepof culturing a microorganism capable of producing thepolyhydroxyalkanoate utilizing alkanoate in a medium containing thealkanoate and a saccharide.

According to a further aspect of the present invention, there isprovided a process of producing polyhydroxyalkanoate, comprising a stepof culturing a microorganism capable of producing a polyhydroxyalkanoateutilizing an alkanoate in a medium containing the alkanoate and apolypeptone.

According to a further aspect of the present invention, there isprovided a process of producing polyhydroxyalkanoate comprising a stepof culturing a microorganism capable of producing a polyhydroxyalkanoateutilizing an alkanoate in a medium containing the alkanoate and anorganic acid participating in TCA cycle.

According to a further aspect of the present invention, there isprovided a process of producing polyhydroxyalkanoate, wherein the amicroorganism is cultured in at least two steps: one is in a mediumcontaining an alkanoate and a polypeptone and the subsequent one is in amedium containing the alkanoate and pyruvic acid or salt thereof withnitrogen source limitation.

According to a further aspect of the present invention, there isprovided Pseudomonas cichorii H45, FERM BP-7374.

According to a further aspect of the present invention, there isprovided a novel bacterial strain Pseudomonas cichorii YN2, FERMBP-7375.

According to a further aspect of the present invention, there isprovided a novel bacterial strain Pseudomonas putida P91, FERM BP-7373.

According to a further aspect of the present invention, there isprovided a novel bacterial strain Pseudomonas jessenii P161, FERMBP-7376.

As already stated above, the present invention provides novelpolyhydroxyalkanoates and novel substituted alkanoic acids to be a rawmaterial therefor and novel microorganisms which have ability to produceand accumulate in the cell the novel polyhydroxyalkanoates, and providesmethods for producing the polyhydroxyalkanoates using suchmicroorganism. According to them, the polyhydroxyalkanoates useful asfunctional polymers in which different functional groups are introducedcan be manufactured very efficiently and in high purity, therefore itmay be expected to be applied to each field such as device and medicalmaterials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart which shows the measurement of nuclear magneticresonance spectrum of 3HFPV synthesized in Example 1;

FIG. 2 is a chart which shows the measurement of ¹H nuclear magneticresonance spectrum of PHA obtained in Example 6;

FIG. 3 is a chart which shows the measurement of ¹³C nuclear magneticresonance spectrum of PHA obtained in Example 6;

FIG. 4 is a chart which shows the measurement of nuclear magneticresonance spectrum of 5-(4-fluorophenoxy)valeric acid obtained inExample 7;

FIG. 5 is a chart which shows the Total Ion Chromatography (TIC) ofGC-MS for a methyl esterification compound of a monomer unitconstituting PHA obtained in Example 8;

FIGS. 6A and 6B are charts which show the mass spectra of the peaks onTIC for a methyl esterification compound of a monomer unit constitutingPHA obtained in Example 8;

FIGS. 7A and 7B are charts which show the mass spectra of the peaks onTIC for a methyl esterification compound of a monomer unit constitutingPHA obtained in Example 8;

FIG. 8 is a chart which shows TIC for a methyl esterification compoundof a monomer unit constituting PHA obtained in Example 9;

FIG. 9 is a chart which shows TIC for a methyl esterification compoundof a monomer unit constituting PHA obtained in Example 10;

FIG. 10 is a chart which shows TIC for a methyl esterification compoundof a monomer unit constituting PHA obtained in Example 11;

FIGS. 11A and 11B are charts which show the Total Ion Chromatography(TIC) of 5-(4-trifluoromethylphenyl)valeric acid (FIG. 11A) and its massspectrum (FIG. 11B);

FIGS. 12A and 12B are charts which show the analytical results of themethylated compounds of PHA copolymers obtained in Example 14. FIG. 12Ais the Total Ion Chromatography (TIC) of the methylated compounds of PHAcopolymers and FIG. 12B is the mass spectrum for the peak (around 36.5min.) containing 3-hydroxy-5-(4-trifluoromethylphenyl)valeric acid whichis an objective unit on the TIC;

FIGS. 13A and 13B are charts which show the analytical results of themethylated compounds of PHA copolymers obtained in Example 15. FIG. 13Ais the Total Ion Chromatography (TIC) of the methylated compounds of PHAcopolymers and FIG. 13B is the mass spectrum for the peak (around 36.5min.) containing 3-hydroxy-5-(4-trifluoromethylphenyl)valeric acid whichis an objective unit on the TIC;

FIG. 14 is a chart which shows the ¹H-NMR spectrum of PHA obtained inExample 30;

FIG. 15 is a chart which shows the ¹H-NMR spectrum of PHA obtained inExample 34;

FIG. 16 is a chart which shows the ¹H-NMR spectrum of4-(4-fluorophenoxy)butyric acid;

FIG. 17 is a chart which shows the ¹³C-NMR spectrum of4-(4-fluorophenoxy)butyric acid;

FIG. 18 are charts which show the GC-MS spectrum data measured aftermethanolysis of PHA recovered from the cultured cells of strain YN2 inExample 41;

FIG. 19 is a chart which shows the ¹H-NMR spectrum of PHA recovered fromthe cultured microbial cells of strain YN2 in Example 41;

FIG. 20 is a chart which shows the ¹H-NMR spectrum of4-(3-fluorophenoxy)butyric acid;

FIG. 21 is a chart which shows the ¹³C-NMR spectrum of4-(3-fluorophenoxy)butyric acid;

FIG. 22 are charts which show the GC-MS spectrum data measured aftermethanolysis of PHA recovered from the cultured microbial cells ofstrain YN2 in Example 47;

FIG. 23 is a chart which shows the ¹H-NMR spectrum of PHA recovered fromthe cultured microbial cells of strain YN2 in Example 47;

FIG. 24 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 53;

FIG. 25 is a chart which shows the mass spectrum of methyl3-hydroxy-5-(4-fluorophenoxy)valerate obtained from the GC-MSmeasurement in Example 53;

FIG. 26 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 54;

FIG. 27 is a chart which shows the mass spectrum of methyl3-hydroxy-5-(4-fluorophenyl)valerate obtained from the GC-MS measurementin Example 54;

FIG. 28 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 55;

FIG. 29 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 55;

FIG. 30 is a chart which shows the mass spectrum of methyl3-hydroxy-5-(4-fluorophenoxy)valerate (3HFPxV) obtained from the GC-MSmeasurement in Example 55;

FIG. 31 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 56;

FIG. 32 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 56;

FIG. 33 is a chart which shows the mass spectrum of methyl3-hydroxy-5-(4-fluorophenyl)valerate (3HFPV) obtained from the GC-MSmeasurement in Example 56;

FIG. 34 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 57;

FIG. 35 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 57;

FIG. 36 is a chart which shows the mass spectrum of methyl3-hydroxy-5-(4-fluorophenyl)valerate (3HFPV) obtained from the GC-MSmeasurement in Example 57;

FIG. 37 is a chart which shows the mass spectrum of methyl3-hydroxy-5-(4-fluorophenoxy)valerate (3HFPxV) obtained from the GC-MSmeasurement in Example 57;

FIG. 38 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 58;

FIG. 39 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 58;

FIG. 40 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 58;

FIG. 41 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 58;

FIG. 42 is a chart which shows the mass spectrum of methyl3-hydroxy-7-phenoxyheptanoate (3HPxHp) obtained from the GC-MSmeasurement in Example 58;

FIG. 43 is a chart which shows the mass spectrum of methyl3-hydroxy-9-phenoxynonanoate (3HPxN) obtained from the GC-MS measurementin Example 58;

FIG. 44 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 59;

FIG. 45 is a chart which shows the mass spectrum of methyl3-hydroxyhexanoate obtained from the GC-MS measurement in Example 59;

FIG. 46 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 59.

FIG. 47 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 59;

FIG. 48 is a chart which shows the mass spectrum of methyl3-hydroxydodecanoate obtained from the GC-MS measurement in Example 59;

FIG. 49 is a chart which shows the mass spectrum of methyl3-hydroxydodecenoate obtained from the GC-MS measurement in Example 59;

FIG. 50 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 59;

FIG. 51 is a chart which shows the mass spectrum of methyl3-hydroxy-7-phenoxyheptanoate (3HPxHp) obtained from the GC-MSmeasurement in Example 59;

FIG. 52 is a chart which shows the mass spectrum of methyl3-hydroxy-9-phenoxynonanoate (3HPxN) obtained from the GC-MS measurementin Example 59;

FIG. 53 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 60;

FIG. 54 is a chart which shows the mass spectrum of methyl3-hydroxy-hexanoate obtained from the GC-MS measurement in Example 60;

FIG. 55 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 60;

FIG. 56 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 60;

FIG. 57 is a chart which shows the mass spectrum of methyl3-hydroxydodecanoate obtained from the GC-MS measurement in Example 60;

FIG. 58 is a chart which shows the mass spectrum of methyl3-hydroxydodecenoate obtained from the GC-MS measurement in Example 60;

FIG. 59 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 60;

FIG. 60 is a chart which shows the mass spectrum of methyl3-hydroxy-7-phenoxyheptanoate (3HPxHp) obtained from the GC-MSmeasurement in Example 60;

FIG. 61 is a chart which shows the mass spectrum of methyl3-hydroxy-9-phenoxynonanoate (3HPxN) obtained from the GC-MS measurementin Example 60;

FIG. 62 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 61;

FIG. 63 is a chart which shows the mass spectrum of methyl3-hydroxy-4-phenoxybutyrate (3HPxB) obtained from the GC-MS measurementin Example 61;

FIG. 64 is a chart which shows the mass spectrum of methyl3-hydroxy-6-phenoxyhexanoate (3HPxHx) obtained from the GC-MSmeasurement in Example 61;

FIG. 65 is a chart which shows the mass spectrum of methyl3-hydroxy-8-phenoxyoctanoate (3HPxO) obtained from the GC-MS measurementin Example 61;

FIG. 66 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 62;

FIG. 67 is a chart which shows the mass spectrum of methyl3-hydroxy-4-phenoxybutyrate (3HPxB) obtained from the GC-MS measurementin Example 62;

FIG. 68 is a chart which shows the mass spectrum of methyl3-hydroxy-6-phenoxyhexanoate (3HPxHx) obtained from the GC-MSmeasurement in Example 62;

FIG. 69 is a chart which shows the mass spectrum of methyl3-hydroxy-8-phenoxyoctanoate (3HPxO) obtained from the GC-MS measurementin Example 62;

FIG. 70 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 63;

FIG. 71 is a chart which shows the mass spectrum of methyl3-hydroxy-octanoate obtained from the GC-MS measurement in Example 63;

FIG. 72 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 63;

FIG. 73 is a chart which shows the mass spectrum of methyl3-hydroxy-4-phenoxybutyrate (3HPxB) obtained from the GC-MS measurementin Example 63;

FIG. 74 is a chart which shows the mass spectrum of methyl3-hydroxy-6-phenoxyhexanoate (3HPxHx) obtained from the GC-MSmeasurement in Example 63;

FIG. 75 is a chart which shows the mass spectrum of methyl3-hydroxy-8-phenoxyoctanoate (3HPxO) obtained from the GC-MS measurementin Example 63;

FIG. 76 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 64;

FIG. 77 is a chart which shows the mass spectrum of methyl3-hydroxyhexanoate obtained from the GC-MS measurement in Example 64;

FIG. 78 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 64;

FIG. 79 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 64;

FIG. 80 is a chart which shows the mass spectrum of methyl3-hydroxydodecanoate obtained from the GC-MS measurement in Example 64;

FIG. 81 is a chart which shows the mass spectrum of methyl3-hydroxydodecenoate obtained from the GC-MS measurement in Example 64;

FIG. 82 is a chart which shows the mass spectrum of methyl3-hydroxy-4-phenoxybutyrate (3HPxB) obtained from the GC-MS measurementin Example 64;

FIG. 83 is a chart which shows the mass spectrum of methyl3-hydroxy-6-phenoxyhexanoate (3HPxHx) obtained from the GC-MSmeasurement in Example 64;

FIG. 84 is a chart which shows the mass spectrum of methyl3-hydroxy-8-phenoxyoctanoate (3HPxO) obtained from the GC-MS measurementin Example 64;

FIG. 85 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 65;

FIG. 86 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 65;

FIG. 87 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 65;

FIG. 88 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 65;

FIG. 89 is a chart which shows the mass spectrum of methyl3-hydroxy-7-phenoxyheptanoate (3HPxHp) obtained from the GC-MSmeasurement in Example 65;

FIG. 90 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 66;

FIG. 91 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 66;

FIG. 92 is a chart which shows the mass spectrum of methyl3-hydroxy-7-phenoxyheptanoate (3HPxHp) obtained from the GC-MSmeasurement in Example 66;

FIG. 93 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 67;

FIG. 94 is a chart which shows the mass spectrum of methyl3-hydroxyhexanoate obtained from the GC-MS measurement in Example 67;

FIG. 95 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 67;

FIG. 96 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 67;

FIG. 97 is a chart which shows the mass spectrum of methyl3-hydroxydodecanoate obtained from the GC-MS measurement in Example 67;

FIG. 98 is a chart which shows the mass spectrum of methyl3-hydroxydodecenoate obtained from the GC-MS measurement in Example 67;

FIG. 99 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 67;

FIG. 100 is a chart which shows the mass spectrum of methyl3-hydroxy-7-phenoxyheptanoate (3HPxHp) obtained from the GC-MSmeasurement in Example 67;

FIG. 101 is a chart which shows the mass spectrum of methyl3-hydroxyhexanoate obtained from the GC-MS measurement in Example 68;

FIG. 102 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 68;

FIG. 103 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 68;

FIG. 104 is a chart which shows the mass spectrum of methyl3-hydroxydodecanoate obtained from the GC-MS measurement in Example 68;

FIG. 105 is a chart which shows the mass spectrum of methyl3-hydroxydodecenoate obtained from the GC-MS measurement in Example 68;

FIG. 106 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 68;

FIG. 107 is a chart which shows the mass spectrum of methyl3-hydroxy-7-phenoxyheptanoate (3HPxHp) obtained from the GC-MSmeasurement in Example 68;

FIG. 108 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 69;

FIG. 109 is a chart which shows the mass spectrum of methyl3-hydroxyhexanoate obtained from the GC-MS measurement in Example 69;

FIG. 110 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 69;

FIG. 111 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 69;

FIG. 112 is a chart which shows the mass spectrum of methyl3-hydroxydodecanoate obtained from the GC-MS measurement in Example 69;

FIG. 113 is a chart which shows the mass spectrum of methyl3-hydroxydodecenoate obtained from the GC-MS measurement in Example 69;

FIG. 114 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 69;

FIG. 115 is a chart which shows the mass spectrum of methyl3-hydroxyhexanoate obtained from the GC-MS measurement in Example 70;

FIG. 116 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 70;

FIG. 117 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 70;

FIG. 118 is a chart which shows the mass spectrum of methyl3-hydroxydodecanoate obtained from the GC-MS measurement in Example 70;

FIG. 119 is a chart which shows the mass spectrum of methyl3-hydroxydodecenoate obtained from the GC-MS measurement in Example 70;

FIG. 120 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenoxyvalerate (3HPxV) obtained from the GC-MS measurementin Example 70;

FIG. 121 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 71;

FIG. 122 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 71;

FIG. 123 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenylvalerate (3HPV) obtained from the GC-MS measurement inExample 71;

FIG. 124 is a chart which shows the mass spectrum of methyl3-hydroxy-octanoate obtained from the GC-MS measurement in Example 72;

FIG. 125 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenylvalerate (3HPV) obtained from the GC-MS measurement inExample 72;

FIG. 126 is a chart which shows the mass spectrum of methyl3-hydroxybutyrate obtained from the GC-MS measurement in Example 73;

FIG. 127 is a chart which shows the mass spectrum of methyl3-hydroxyoctanoate obtained from the GC-MS measurement in Example 73;

FIG. 128 is a chart which shows the mass spectrum of methyl3-hydroxydecanoate obtained from the GC-MS measurement in Example 73;and

FIG. 129 is a chart which shows the mass spectrum of methyl3-hydroxy-5-phenylvalerate (3HPV) obtained from the GC-MS measurement inExample 73.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For solving the above described problems, the inventor et al.strenuously carried out researches for search of innovativemicroorganisms capable of producing PHA and accumulating the same in thecells and a method of manufacturing desired PHA using the innovativemicroorganism, particularly with the aim of developing PHA havingsubstituted or unsubstituted phenoxy groups, phenyl groups andcyclohexyl groups on the side chain, which is useful as device materialsand medical materials, and completed the invention.

The polyhydroxyalkanoate of the present invention is characterized byhaving a monomer unit composition represented by formula (1).

A_(m)B_((1−m))  (1)

(wherein A is represented by formula (2), B is at least one or moreselected from monomer units represented by formula (3) or (4), and m is0.01 or more and less than 1).

(In formulae, n is 0 to 10, k is 3 or 5, and R is at least one or moregroups selected from groups represented by formulae (5) to (7)).

(In formula (5), R1 is a group selected from hydrogen atom (H), halogenatom, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇, and q is selected from integersof 1 to 8; In formula (6), R2 is a group selected from hydrogen atom(H), halogen atom, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇, and r is selectedfrom integers of 1 to 8; In formula (7), R3 is a group selected fromhydrogen atom (H), halogen atom, —CN, —NO₂, —CF₃, —C₂F₃, and —C₃F₇, ands is selected from integers of 1 to 8;

wherein, when one kind of group is selected,

as R in formula (2),

the group of Ri=H and q=2, the group of R1=H and q=3, and the group ofRi=—NO₂ and q=2 in formula (5),

the group of R2=halogen atom and r=2 [however, only when two componentsare selected from formula (3) or (4) as the above described B], thegroup of R2=—CN and r=3, and the group of R2=—NO₂ and r=3 in formula(6), and

the group of R3=H and s=1 and the group of R3=H and s=2 in formula (7)

are excluded from alternatives, and

when two kinds of groups are selected, in formula (6), a combination oftwo kinds of groups of R2=halogen atom and r=2 and 4 [however, only whenone component is selected from formula (3) or (4) as the above describedB]

are excluded from alternatives).

Herein, the polyhydroxyalkanoate of the present invention may includemore than two kinds of monomer units represented by formula (2), but itis preferably designed so that the appropriate number of monomer unitsare included, considering the needed polymer's functionality andproperties. Generally, when alkanoates up to about five kinds are usedas a raw material of desired monomer units, “secondary” substrates withthe chain length sequentially decreased by two methylene units are newlyproduced by β oxidation from part of the alkanoates, as describedpreviously, and captured as monomer units of the PHA. Thus, about tenkinds or less of monomer units represented by formula (2) are includedin PHA and it is expected that the object of the present invention besufficiently achieved. Furthermore, if fine control of the functionalityand the property is desired, configuration with more species of monomerunits is also possible.

Also, with respect to substituted positions of R1, R2 and R3, for any ofa ortho, meta or para position, and for a first position in the case ofthe cyclohexyl group of R3, polyhydroxyalkanoate containingcorresponding monomer units can be configured, but if there is nosignificant differences in functionality and properties for any isomer,it is advantageously configured with constituents at the meta or paraposition in terms of yields or ease with which it is captured in thepolymer.

Also, the method of manufacturing polyhydroxyalkanoate of the presentinvention is a method of manufacturing polyhydroxyalkanoate havingmonomer unit composition represented by formula (1) usingmicroorganisms, characterized in that microorganisms are culturedtogether with the alkanoate and the polyhydroxyalkanoate is extractedfrom the cells of organisms to obtain the polyhydroxyalkanoate havingmonomer unit composition represented by formula (1).

A_(m)B_((1−m))  (1)

(wherein A is represented by formula (2), B is at least one or moreselected from monomer units represented by formula (3) or (4), and m is0.01 or more and less than 1).

(In formulae, n is 0 to 10, k is 3 or 5, and R is at least one or moregroups selected from groups represented by formulae (5) to (7)).

(In formula (5), R1 is a group selected from hydrogen atom (H), halogenatom, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇, and q is selected from integersof 1 to 8;

In formula (6), R2 is a group selected from hydrogen atom (H), halogenatom, —CN, —NO₂, —CF₃, —C2F₅ and —C₃F₇, and r is selected from integersof 1 to 8;

In formula (7), R3 is a group selected from hydrogen atom (H), halogenatom, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇, and s is selected from integersof 1 to 8;

wherein, when one kind of group is selected, as R in formula (2),

the group of R1=H and q=2, the group of R1=H and q=3, and the group ofR1=—NO₂ and q=2 in formula (5),

the group of R2=halogen atom and r=2, the group of R2=—CN and r=3, andthe group of R2=—NO₂ and r=3 in formula (6), and

the group of R3=H and s=1 and the group of R3=H and s=2 in formula (7)

are excluded from alternatives, and

when two kinds of groups are selected, in formula (6), a combination oftwo kinds of groups of R2=halogen atom and r=2

are excluded from alternatives).

Herein, the polyhydroxyalkanoate of the present invention may more thantwo kinds of monomer units represented by formula (2), but it ispreferably synthesized so that the appropriate number of monomer unitsare included, considering the needed polymer's functionality andproperties. Generally, when alkanoates of up to about five kinds areused as a raw material for desired substrates, “secondary” substrateswith the chain length shortened by two methylene units are newlyproduced by β oxidation from the alkanoates as described previously, andcaptured as monomer units in PHA. Thus, monomer units of up to ten kindsrepresented by formula (2) are included in PHA, and it is expected thatthe object of the present invention is sufficiently achieved.Furthermore, if fine control of the functionality and the property isdesired, culture may be performed to include more species of monomerunits.

Also, with respect to substituted positions of R1, R2 and R3, for any ofa ortho, meta or para position, and for a first position in the case ofthe cyclohexyl group of R3, polyhydroxyalkanoate containingcorresponding monomer units can be configured, but if there is nosignificant differences in functionality and properties for any isomer,it is advantageously configured with constituents at the meta or paraposition in terms of yields or ease with which it is captured in thepolymer.

Furthermore, the inventors did strenuous research to develop a methodfor obtaining desired PHA having little or no undesired monomer unitscoexisting therein, and as a result, found that when the microorganismis cultured in a culture medium containing as the sole carbon source thealkanoate to be the raw material for PHA and a saccharide, it ispossible to produce PHA having little or no undesired monomer unitscoexisting therein, leading to the completion of the invention.

That is, the method of manufacturing polyhydroxyalkanoate (PHA) of thepresent invention is a method of manufacturing polyhydroxyalkanoatecontaining monomer units represented by formula (23) usingmicroorganisms, characterized by having a process in which amicroorganism capable of synthesizing polyhydroxyalkanoate containingmonomer units represented by formula (23) from the alkanoate representedby general formula (22) are cultured in a culture medium containing asthe sole carbon source the alkanoate of formula (22). and a saccharide.

(In above formulae, R is at least one or more groups represented byformula (24), and R′ is one or more groups selected from the groupsselected in formula (22), the group of t-2 in the selected groups, thegroup of t-4 in the selected groups and the group of t-6 in the selectedgroups. Herein, t-2, t-4 and t-6 can be only integers of 1 or more.)

(In the above formula, R4 represents a saturated or unsaturated phenylgroup, a saturated or unsaturated phenoxy group, and a saturated orunsaturated cyclohexyl group, and t represents an integer in the rangeof 1 to 8 independently.)

Herein, more than one kinds of alkanoate represented by formula (22) maybe used when culture is carried out, but the appropriate number thereofare preferably used, considering the needed polymer's functionality andproperties. Generally, when alkanoates of up to five kinds representedby formula (22) are used as raw materials for the desired monomer units,“secondary” substrates with the chain length shortened by two methyleneunits are newly produced by β oxidation from part of the alkanoates asdescribed previously, and captured as monomer units of PHA. Thus,monomer units of up to ten kinds represented by formula (2), forexample, are included in PHA, and it is expected that the abovedescribed object be sufficiently achieved. Furthermore, if fine controlof the functionality and the property is desired, more kinds ofalkanoates can be used.

Substituents at the group of R4 described above include halogen atom,—CN, —NO₂, —CF₃, —C₂F₅, —C₃F₇ and the like. With respect to substitutedpositions of R4, in any of a ortho, meta or para position, and in afirst position in the case of the cyclohexyl group, polyhydroxyalkanoateconstituted by corresponding monomer units can be obtained, but if thereis no significant differences in functionality and properties for anyisomer, constituents at the meta or para position can be suitably usedin terms of yields or ease with which it is captured in the polymer.

Also, for saccharides, for example, glucose, fructose, mannose and thelike may be suitably used.

Furthermore, the inventors did strenuous research to develop a methodfor producing desired PHA having little or no undesired monomer unitscoexisting therein, and as a result, found that when the microorganismis cultured in a culture medium containing as the sole carbon source analkanoate to be the raw material for PHA and polypeptone only, it ispossible to produce PHA having little or no undesired monomer unitscoexisting therein, leading to the completion of the invention.

That is, the method of manufacturing polyhydroxyalkanoate (PHA) of thepresent invention is a method of manufacturing polyhydroxyalkanoatecontaining monomer units represented by formula (23) usingmicroorganisms, characterized by having a process in whichmicroorganisms capable of synthesizing polyhydroxyalkanoate containingmonomer units represented by formula (23) from the alkanoate representedby formula (22) are cultured in a culture medium containing thealkanoate and polypeptone as the only carbon source.

(In the above described formulae, R is at least one or more groupsrepresented by formula (24), and R′ is one or more groups selected fromthe groups selected in formula (22), the group of t-2 in the selectedgroups, the group of t-4 in the selected groups and the group of t-6 inthe selected groups. Herein, t-2, t-4 and t-6 can be only integers of 1or more.)

(In the above described formula, R4 represents a saturated orunsaturated phenyl group, a saturated or unsaturated phenoxy group, anda saturated or unsaturated cyclohexyl group, and t represents an integerin the range of 1 to 8 independently.)

Herein, more than one kinds of alkanoate represented by formula (22) maybe used when culture is carried out, but the appropriate number thereofare preferably used, considering the needed polymer's functionality andproperties. Generally, when up to five kinds of alkanoates representedby formula (22) are used as raw materials for the desired monomer units,secondary” substrates with the chain length shortened by two methyleneunits are newly produced by β oxidation from a part of the alkanoates asdescribed previously, and captured as the monomer units in PHA. Thus, upto about ten kinds of monomer units represented by formula (2), forexample, are included in PHA, and it is expected that the abovedescribed object be sufficiently achieved. Furthermore, if fine controlof the functionality and the property is desired, more kinds ofalkanoate can be used.

Substituents at the group of R4 described above include halogen atom,—CN, —NO₂, —CF₃, —C₂F₅, —C₃F₇ and the like. With respect to substitutedpositions of R4, in any of a ortho, meta or para position, and in afirst position in the case of the cyclohexyl group, polyhydroxyalkanoateconstituted by corresponding monomer units can be obtained, but if thereis no significant differences in functionality and properties for anyisomer, constituents at the meta or para position can be suitably usedin terms of yields or ease with which it is captured in the polymer.

Furthermore, the inventors did strenuous research to develop a methodfor efficiently producing desired PHA having little or no undesiredmonomer units coexisting therein, and as a result, found that when themicroorganism are cultured in a culture medium containing as the solecarbon source an alkanoate to be the raw material for PHA and an organicacid associated with the TCA cycle, it is possible to produce PHA havinglittle or no undesired monomer units coexisting therein, leading to thecompletion of the invention.

That is, the method of producing polyhydroxyalkanoate (PHA) of thepresent invention is a method of producing polyhydroxyalkanoatecontaining monomer units represented by formula (23) usingmicroorganisms, characterized by having a process in whichmicroorganisms capable of synthesizing polyhydroxyalkanoate containingmonomer units represented by formula (23) from the alkanoate representedby the following general formula (22) are cultured in a culture mediumincluding the alkanoate and only an organic acid associated with the TCAcycle as a carbon source other than the alkanoate represented by thefollowing formula (22).

(In the above described formulae, R is at least one or more groupsrepresented by formula (24), and R′ is one or more groups selected fromthe groups selected in formula (22), the group of t-2 in the selectedgroups, the group of t-4 in the selected groups and the group of t-6 inthe selected groups. Herein, t-2, t-4 and t-6 can be only integers of 1or more.)

(In the above described formula, R4 represents a saturated orunsaturated phenyl group, a saturated or unsaturated phenoxy group, anda saturated or unsaturated cyclohexyl group, and t represents an integerin the range of 1 to 8 independently.)

Herein, more than one kinds of alkanoate represented by formula (22) maybe used when culture is carried out, but the appropriate number thereofare preferably used, considering the needed polymer's functionality andproperties. Generally, when up to about five kinds of alkanoatesrepresented by formula (22) are used as the raw materials, “secondary”substrates with the chain length shortened by two methylene units arenewly produced by β oxidation from a part of the alkanoates as describedpreviously, and captured as the monomer units of PHA. Thus, up to aboutten kinds of monomer units, for example, as represented by formula (2),are included in PHA, and it is expected that the above described objectbe sufficiently achieved. Furthermore, if fine control of thefunctionality and the property is desired, more kinds of alkanoates canbe used.

Substituents at the group of R4 described above include halogen atom,—CN, —NO₂, —CF₃, —C₂F₅, —C₃F₇ and the like. With respect to substitutedpositions of R4, in any of a ortho, meta or para position, and in afirst position in the case of the cyclohexyl group, polyhydroxyalkanoateconstituted by corresponding monomer units can be obtained, but if thereis no significant differences in functionality and properties for anyisomer, constituents at the meta or para position can be suitably usedin terms of yields or ease with which it is captured in the polymer.

Also, for organic acids associated with the TCA cycle, organic acidsexisting in the TCA cycle itself, for example citric acid, succinicacid, fumaric acid, malic acid and salts thereof, and organic acidsexisting on the main flux to the TCA cycle, for example lactic acid,pyruvic acid and salts thereof may be suitably used.

Furthermore, the inventors did strenuous research to develop a methodfor obtaining desired PHA having little or no undesired monomer unitscoexisting therein efficiently, and as a result, found that whenmicroorganisms are cultured in at least two steps: first in a mediumcontaining as the sole carbon source an alkanoate to be a raw materialfor PHA and polypeptone, and then in a medium containing the alkanoateand pyruvic acid or salts thereof as the sole carbon source withnitrogen limitation, it is possible to produce PHA having little or noundesired monomer units coexisting therein.

That is, the method of producing polyhydroxyalkanoate (PHA) of thepresent invention is a method of manufacturing polyhydroxyalkanoatecontaining monomer units represented by formula (23) usingmicroorganisms, characterized by having a process in whichmicroorganisms capable of synthesizing polyhydroxyalkanoate containingmonomer units represented by formula (23) from the alkanoate representedby the following general formula (22) are cultured in at least twosteps: first in a culture medium containing as the sole carbon sourcethe alkanoate represented by formula (22) and polypeptone, and then in aculture medium containing as the sole carbon source the alkanoaterepresented by formula (22) and pyruvic acid or salts thereof undernitrogen limitation.

(In the above described formulae, R is at least one or more groupsrepresented by formula (24), and R′ is one or more groups selected fromthe groups selected in the above described formula (22), the group oft-2 in the selected groups, the group of t-4 in the selected groups andthe group of t-6 in the selected groups. Herein, t-2, t-4 and t-6 can beonly integers of 1 or more.)

(In the above described formula, R4 represents a saturated orunsaturated phenyl group, a saturated or unsaturated phenoxy group, anda saturated or unsaturated cyclohexyl group, and t represents an integerin the range of 1 to 8 independently.)

Herein, more than one kinds of alkanoate represented by formula (22) maybe used when culture is carried out, but the appropriate number thereofare preferably used, considering the needed polymer's functionality andproperties. Generally, when up to about five kinds of alkanoatesrepresented by formula (22) are used as the raw materials, “secondary”substrates shortened by two methylene units are newly produced by βoxidation from part of the alkanoates as described previously, andcaptured as the monomer units in PHA, up to about ten kinds of monomerunits, for example, those represented by formula (2), are included inPHA, and it is expected that the above described object be sufficientlyachieved. Furthermore, if fine control of the functionality and theproperty is desired, more kinds of alkanoate can be used.

Substituents at the group of R4 described above include halogen atom,—CN, —NO₂, —CF₃, —C₂F₅, —C₃F₇ and the like. With respect to substitutedpositions of R4, in any of a ortho, meta or para position, and in afirst position in the case of the cyclohexyl group, polyhydroxyalkanoateconstituted by corresponding monomer units can be obtained, but if thereis no significant differences in functionality and properties for anyisomer, constituents at the meta or para position can be suitably usedin terms of yields or ease with which it is captured in the polymer.

Also, new strains related to the present invention are characterized byhaving synthetic systems of polyhydroxyalkanoate including alkanoaterepresented by formula (22) to monomer units represented by formula(23).

(In the above described formulae, R is at least one or more groupsrepresented by formula (24), and R′ is one or more groups selected fromthe groups selected in the above described formula (22), the group oft-2 in the selected groups, the group of t-4 in the selected groups andthe group of t-6 in the selected groups. Herein, t-2, t-4 and t-6 can beonly integers of 1 or more.)

(wherein R4 is a substituted or unsubstituted phenyl group, asubstituted or unsubstituted phenoxy group, or a substituted orunsubstituted cyclohexyl group; and t is independently an integer of1-8).

A substituent in the above described R4 group includes a halogen atom,—CN, —NO₂, —CF₃, —C₂F₅, —C₃F₇ or the like.

Hereinafter, a new polyhydroxyalkanoate of the present invention will beillustrated.

A new polyhydroxyalkanoate of the present invention is the one offormula (8):

which contains 3-hydroxy-5-(4-fluorophenyl)valeric acid as a monomerunit.

Further, the polyhydroxyalkanoate is of formula (9):

which contains 3-hydroxy-5-(4-trifluoromethylphenyl)valeric acid as amonomer unit.

In addition, a material substrate when producing new PHA of the presentinvention using microorganisms is 5-(4-trifluoromethylphenyl)valericacid of the following chemical formula (21):

and the material substrate itself is a new compound.

Further, the polyhydroxyalkanoate is of formula (10):

which contains 3-hydroxy-4-(4-nitrophenoxy)butyric acid as a monomerunit.

Further, the polyhydroxyalkanoate is of formula (11):

which contains 3-hydroxy-4-(4-cyanophenoxy)butyric acid as a monomerunit.

Further, the polyhydroxyalkanoate is of formula (12):

which contains 3-hydroxy-4-(4-fluorophenoxy)butyric acid as a monomerunit.

Further, the polyhydroxyalkanoate is of formula (13):

which contains 3-hydroxy-4-(3-fluorophenoxy)butyric acid as a monomerunit.

Further, the polyhydroxyalkanoate is of formula (14):

which contains 3-hydroxy-4-phenoxybutyric acid as a monomer unit.Herein, as the monomer unit except 3-hydroxy-4-phenoxybutyric acid offormula (14), at least one or more of the monomer units of formula (3)or (4) are contained.

(wherein n is 0-10)(wherein k is 3 or 5)

Further, the polyhydroxyalkanoate is of formula (15):

which contains 3-hydroxy-5-phenoxyvaleric acid as a monomer unit.Herein, as the monomer unit except 3-hydroxy-5-phenoxyvaleric acid offormula (15), at least one or more of the monomer units of formula (3)or (4) are contained.

(wherein n is 0-10)(wherein k is 3 or 5)

Further, the polyhydroxyalkanoate is of formula (16):

which contains 3-hydroxy-5-(4-fluorophenoxy)valeric acid as a monomerunit. Herein, as the monomer unit except3-hydroxy-5-(4-fluorophenoxy)valeric acid of formula (16), thepolyhydroxyalkanoate contains at least one or more of the monomer unitsof formula (3) or (4) and excludes the monomer unit of three componentsystem.

(wherein n is 0-10) (wherein k is 3 or 5)

Further, the polyhydroxyalkanoates are of formulas (8) and (16):

which contain 3-hydroxy-5-(4-fluorophenyl)valeric acid and3-hydroxy-5-(4-fluorophenoxy)valeric acid as monomer units.

Further, the polyhydroxyalkanoates are of formulas (15) and (17):

which contain 3-hydroxy-5-phenoxyvaleric acid and3-hydroxy-7-phenoxyheptanoic acid as monomer units. Herein, as themonomer unit except 3-hydroxy-5-phenoxyvaleric acid and3-hydroxy-7-phenoxyheptanoic acid of formulas (15) and (17), at leastone or more of the monomer units of formula (3) or (4) are contained.

(wherein n is 0-10) (wherein k is 3 or 5)

Further, the polyhydroxyalkanoates are of formulas (14), (18) and (19):

which contain 3-hydroxy-4-phenoxybutyric acid,3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-8-phenoxyoctanoic acid asmonomer units. Herein, as the monomer unit except3-hydroxy-4-phenoxybutyric acid, 3-hydroxy-6-phenoxyhexanoic acid and3-hydroxy-8-phenoxyoctanoic acid of formulas (14), (18) and (19), atleast one or more of the monomer units of formula (3) or (4) arecontained.

(wherein n is 0-10) (wherein k is 3 or 5)

Further, the polyhydroxyalkanoates are of formulas (15), (17) and (20):

which contain 3-hydroxy-5-phenoxyvaleric acid,3-hydroxy-7-phenoxyheptanoic acid and 3-hydroxy-9-phenoxynonanoic acidas monomer units. Herein, as the monomer unit except3-hydroxy-5-phenoxyvaleric acid, 3-hydroxy-7-phenoxyheptanoic acid and3-hydroxy-9-phenoxynonanoic acid of formulas (15), (17) and (20), atleast one or more of the monomer units of formula (3) or (4) arecontained.

(wherein n is 0-10) (wherein k is 3 or 5)

Hereinafter, a manufacturing method for polyhydroxyalkanoates will beillustrated.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) of formula (8) by beingcultivated in a culture medium containing 5-(4-fluorophenyl)valeric acid(FPVA) of formula (25).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having a step of cultivatingmicroorganisms which produce polyhydroxyalkanoates containing themonomer unit of 3HFPV of formula (8) using FPVA in a culture mediumcontaining FPVA of formula (25).

In addition, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining FPVA of formula (25) and saccharides.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining FPVA of formula (25) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining FPVA of formula (25) and organic acids associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 stepculturing: first in a culture medium containing FPVA of formula (25) andpolypeptone, and then in a culture medium containing FPVA of formula(25) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-5-(4-trifluoromethylphenyl)valeric acid (3HCF₃PV) of formula(9) by being cultured in a culture medium containing5-(4-trifluoromethylphenyl)valeric acid (CF₃PVA) of formula (21).

A manufacturing method for polyhydroxyalkanoates of the presentinvention comprise is characterized by culturing the microorganism whichcan produce polyhydroxyalkanoates containing the monomer unit of 3HCF₃PVof formula (9) from CF₃PVA of formula (21) in a culture mediumcontaining CF₃PVA.

In addition, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining CF₃PVA of formula (21) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining CF₃PVA of formula (21) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining CF₃PVA of formula (21) and an organic acid associated withthe TCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least two stepculturing: first in a culture medium containing CF₃PVA of formula (21)and polypeptone, and then in a culture medium containing CF₃PVA offormula (21) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-4-(4-nitrophenoxy)butyric acid (3HNO₂PxB) formula (10) bybeing cultured in a culture medium containing 4-(4-nitrophenoxy)butyricacid (NO₂PxBA) of formula (26).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by culturing a microorganism which canproduce polyhydroxyalkanoates containing the monomer unit of 3HNO₂PxBrepresented by formula (10) using NO₂PxBA in a culture medium containingNO₂PxBA of formula (26).

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining NO₂PxBA of formula (26) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining NO₂PxBA of formula (26) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining NO₂PxBA of formula (26) and an organic acid associated withthe TCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganism is performed by at least 2 steps ofculturing, first in a culture medium containing NO₂PxBA of formula (26)and polypeptone, and then in a culture medium containing NO₂PxBA andpyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-4-(4-cyanophenoxy)butyric acid (3HCNPxB) of formula (11) bybeing cultivated in a culture medium containing4-(4-cyanophenoxy)butyric acid (CNPxBA) of formula (27).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having steps of cultivating themicroorganism which can produce polyhydroxyalkanoates containing themonomer unit of 3HCNPxB represented by formula (11) from CNPxBA offormula (27) in a culture medium containing CNPxBA.

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining CNPxBA of formula (27) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining CNPxBA of formula (27) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining CNPxBA of formula (27) and an organic acid associated withthe TCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganism is performed by at least 2 steps ofculturing: first in a culture medium containing CNPxBA of formula (27)and polypeptone, and then in a culture medium containing CNPxBA offormula (27) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-4-(4-fluorophenoxy)butyric acid (3HFPxB) of formula (12) bybeing cultivated in a culture medium containing4-(4-fluorophenoxy)butyric acid (FPxBA) of formula (28).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivating amicroorganism which can produce polyhydroxyalkanoates containing themonomer unit of 3HFPxB of formula (12) using FPxBA in a culture mediumcontaining FPxBA of formula (28).

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining FPxBA of formula (28) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining FPxBA of formula (28) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining FPxBA of formula (28) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing: first in a culture medium containing FPxBA of formula (28)and polypeptone, and then in a culture medium containing FPxBA offormula (28) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-4-(3-fluorophenoxy)butyric acid (3HmFPxB) of formula (13) bybeing cultivated in a culture medium containing4-(3-fluorophenoxy)butyric acid (mFPxBA) of formula (29).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having steps of cultivating microorganismswhich produce polyhydroxyalkanoates containing the monomer unit of3HmFPxB of formula (13) using mFPxBA in a culture medium containingmFPxBA of formula (29).

In addition, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining mFPxBA of formula (29) and saccharides.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining mFPxBA of formula (29) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining mFPxBA of formula (29) and organic acids associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing: first in a culture medium containing mFPxBA of formula (29)and polypeptone, and then in a culture medium containing mFPxBA offormula (29) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-5-phenylvaleric acid (3HPV) of formula (30) by beingcultivated in a culture medium containing 5-phenylvaleric acid (PVA) offormula (31).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer unit of 3HPV of formula (30) using PVA in a culture mediumcontaining PVA of formula (31) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining PVA of formula (31) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganisms is performed in a culture mediumcontaining PVA of formula (31) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganism is performed by at least 2 steps ofculturing: first in a culture medium containing PVA of formula (31) andpolypeptone, and then in a culture medium containing PVA of formula (31)and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-6-phenylhexanoic acid (3HPHx) of formula (32) by beingcultivated in a culture medium containing 6-phenylhexanoic acid (PHxA)of formula (33).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer unit of 3HPHx of formula (32) using PHxA in a culture mediumcontaining PHxA of formula (33) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PHxA of formula (33) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PHxA of formula (33) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganism is performed by at least 2 steps ofculturing: first in a culture medium containing PHXA of formula (33) andpolypeptone, and then in a culture medium containing PHxA of formula(33) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-4-phenoxybutyric acid (3HPxB) of formula (14) by beingcultivated in a culture medium containing 4-phenoxybutyric acid (PxBA)of formula (34).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer unit of 3HPxB of formula (14) using PxBA in a culture mediumcontaining PxBA of formula (34).

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxBA of formula (34) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxBA of formula (34) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxBA of formula (34) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing: first in a culture medium containing PxBA of formula (34) andpolypeptone, and then in a culture medium containing PxBA of formula(34) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-5-phenoxyvaleric acid (3HPxV) of formula (15) by beingcultivated in a culture medium containing 5-phenoxyvaleric acid (PxVA)of formula (35).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer unit of 3HPxV of formula (15) using PxVA in a culture mediumcontaining PxVA of formula (35).

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxVA of formula (35) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxVA of formula (35) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxVA of formula (35) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganism is performed by at least 2 steps ofculturing: first in a culture medium containing PxVA of formula (35) andpolypeptone, and then in a culture medium containing PxVA of formula(35) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-5-(4-fluorophenoxy)valeric acid (3HFPxV) of formula (16) bybeing cultivated in a culture medium containing5-(4-fluorophenoxy)valeric acid (FPxVA) of formula (36).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer unit of 3HFPxV of formula (16) using FPxVA in a culture mediumcontaining FPxVA of formula (36) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining FPxVA of formula (36) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining FPxVA of formula (36) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing, first in a culture medium containing FPxVA of formula (36)and polypeptone, and then in a culture medium containing FPxVA offormula (36) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing a monomer unit of3-hydroxy-4-cyclohexylbutyric acid (3HCHB) of formula (37) by beingcultivated in a culture medium containing 4-cyclohexylbutyric acid(CHBA) of formula (38).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer unit of 3HCHB of formula (37) using CHBA in a culture mediumcontaining CHBA of formula (38) and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining CHBA of formula (38) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining CHBA of formula (38) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganism is performed by at least 2 steps in aculture medium containing CHBA of formula (38) and polypeptone followedby in a culture medium containing CHBA of formula (38) and pyruvic acidor its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing monomer units of3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) and3-hydroxy-5-(4-fluorophenoxy)valeric acid (3HFPxV) of formulas (8) and(16), respectively, by being cultivated in a culture medium containing5-(4-fluorophenyl)valeric acid (FPVA) and 5-(4-fluorophenoxy)valericacid (FPxVA) of formulas (25) and (36), respectively.

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer units of 3HFPV and 3HFPxV of formulas (8) and (16),respectively, using FPVA and FPxVA in a culture medium containing FPVAand FPxVA of formulas (25) and (36), respectively, and a saccharide.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining FPVA and FPxVA of formulas (25) and (36), respectively, andpolypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism. is performed in a culture mediumcontaining FPVA and FPxVA of formulas (25) and (36), respectively, andan organic acid associated with the TCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing; first in a culture medium containing FPVA and FPxVA offormulas (25) and (36), respectively, and polypeptone, and then in aculture medium containing FPVA and FPxVA of formulas (25) and (36),respectively, and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing monomer units of3-hydroxy-5-phenoxyvaleric acid (3HPxV) and 3-hydroxy-7-phenoxyheptanoicacid (3HPxHp) of formulas (15) and (17), respectively, by beingcultivated in a culture medium containing 7-phenoxyheptanoic acid(PxHpA) of formulas (39).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer units of 3HPxV and 3HPxHp of formulas (15) and (17),respectively, using PxHpA in a culture medium containing PxHpA offormula (39) and a saccharide.

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxHpA of formula (39) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxHpA of formula (39) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing; first in a culture medium containing PxHpA of formula (39)and polypeptone, and then in a culture medium containing PxHpA offormula (39) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydroxyalkanoates containing monomer units of3-hydroxy-4-phenoxybutyric acid (3HPxB), 3-hydroxy-6-phenoxyhexanoicacid (3HPxHx) and 3-hydroxy-8-phenoxyoctanoic acid (3HPxO) of formulas(14), (18) and (19), respectively, by being cultivated in a culturemedium containing 8-phenoxyoctanoic acid (PxOA) of formula (40).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer units of 3HPxB, 3HPxHx and 3HPxO of formulas (14), (18) and(19), respectively, using PxOA in a culture medium containing PxOA offormula (40) and a saccharide.

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxOA of formula (40) and polypeptone. Further, anothermanufacturing method is characterized in that cultivation of themicroorganism is cultivated in a culture medium containing PxOA offormula (40) and an organic acid associated with the TCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing: first in a culture medium containing PxOA of formula (40) andpolypeptone, and then in a culture medium containing PxOA of formula(40) and pyruvic acid or its salt with nitrogen limitation.

The present inventors have succeeded in obtaining microorganisms whichcan produce polyhydrbxyalkanoates containing monomer units of3-hydroxy-5-phenoxyvaleric acid (3HPxV), 3-hydroxy-7-phenoxyheptanoicacid (3HPxHp) and 3-hydroxy-9-phenoxynonanoic acid (3HPxN) of formulas(15), (17) and (20), respectively, by being cultivated in a culturemedium containing 11-phenoxyundecanoic acid (PxUDA) of formula (41).

A manufacturing method for polyhydroxyalkanoates of the presentinvention is characterized by having the step of cultivatingmicroorganisms which can produce polyhydroxyalkanoates containing themonomer units of 3HPxV, 3HPxHp and 3HPxN of formulas (15), (17) and(20), respectively, using PxUDA in a culture medium containing PxUDA offormula (41) and a saccharide.

In addition, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxUDA of formula (41) and polypeptone.

Further, another manufacturing method is characterized in thatcultivation of the microorganism is performed in a culture mediumcontaining PxUDA of formula (41) and an organic acid associated with theTCA cycle.

Furthermore, another manufacturing method is characterized in thatcultivation of the microorganisms is performed by at least 2 steps ofculturing; first in a culture medium containing PxUDA of formula (41)and polypeptone, and then in a culture medium containing PxUDA offormula (41) and pyruvic acid or its salt with nitrogen limitation.

Further, four new strains suitably usable for production of the abovedescribed polyhydroxyalkanoates of the present invention includePseudomonas cichorii YN2, FERM BP-7375, Pseudomonas cichorii H45, FERMBP-7374, Pseudomonas putida P91, FERM BP-7373 and Pseudomonas jesseniiP161, FERM BP-7376.

The PHAs according to the present invention are those PHAs which containmonomer units with a variety of structures having substituents useful asdevice and medical materials and others on the side chain, and morespecifically, those which have the above-mentioned substituted ornon-substituted phenoxy, phenyl and cyclohexyl groups on the side chain.In addition, the method according to the present invention enables theproduction of desired PHAs at a high purity and a high yield by usingthe microorganisms. Furthermore, the present invention can providestrains capable of efficient synthesis of the PHAs at a high purity. Ingeneral, the PHAs according to the present invention are of only the Rform, and are isotactic polymers.

<Saccharides and Organic Acids Involved in the TCA Cycle: Differencesfrom Conventional Technology>

One method for producing PHA according to the present invention ischaracterized in that when a microorganism is cultured, in addition toalkanoate for introducing the desired monomer unit, only saccharide(s)or organic acid(s) involved in the TCA cycle as the carbon source otherthan the alkanoate are added into the medium, so that the PHA producedby and accumulated in the microorganism has a significantly high contentof the monomer unit of interest or alternatively only the monomer unitof interest. The effect of facilitating the preference of thisparticular monomer unit is achieved by adding into the medium onlysaccharide(s) or organic acid(s) involved in the TCA cycle as the carbonsource other than the alkanoate.

That is, the inventors have found that when culture is carried out usingsaccharide(s) or organic acid(s) involved in the TCA cycle as theco-existing substrate and together with alkanoate for introducing thedesired monomer unit, the PHA of interest is produced at a particularlysuperior yield and purity, compared to conventional methods employingmcl-alkanoate, such as nonanoate or octanoate, as the co-existingsubstrate, and that this effect is achieved by culture methods allowingacetyl-CoA that is a carbon source as well as an energy source of themicroorganism to be produced by processes independent on theβ-oxidation, and have reached the present invention.

In the methods according to the invention, saccharide compounds, forexample, glucose, fructose, mannose, and the like, are utilized asgrowth substrates for microorganisms, and the produced PHA is ofalkanoate for introducing the desired monomer unit which is existingwith saccharide(s), so that there is contained none of or an extremelysmall amount of monomer units derived from the saccharides such asglucose and others. In this. point, the methods according to the presentinvention is essentially different in structures and effects fromconventional methods for microbial production of PHA in whichsaccharides themselves, such as glucose and others, are employed as theraw substrate of the monomer unit to be introduced into the PHA.

<Polypeptone: Differences from Conventional Technology>

One method for producing PHA according to the present invention ischaracterized in that when a microorganism is cultured, in addition toalkanoate, a raw material, for introducing the desired monomer unit,only polypeptone as the carbon source other than the alkanoate is addedinto the medium, so that the PHA produced by and accumulated in themicroorganism has a significantly high content of the monomer unit ofinterest or alternatively only the monomer unit of interest. The effectof facilitating the preference of this particular monomer unit isachieved by adding into the medium only polypeptone as the carbon sourceother than the alkanoate.

As examples of utilizing polypeptone in microbial production of PHA,Japanese Patent Application Laid-Open Nos. 5-49487, 5-64591, 5-214081,6-145311, 6-284892, 7-48438, 8-89264, 9-191893, and 11-32789 disclosethat when PHA is produced by microorganisms, polypeptone is allowed tobe contained in the media. However, all of these utilize polypeptoneduring pre-culture, that is, at the stage of simply growing cells, andthere are not contained substrates resulting in the monomer unit of PHAduring pre-culture. Furthermore, there are no examples utilizingpolypeptone at the stage of allowing cells to produce PHA. In contrast,the present invention is intended to produce and accumulate PHA, as wellas to grow cells, with alkanoate for introducing the desired monomerunit and by co-existing only polypeptone as the carbon sources otherthan the alkanoate. The production method according to the presentinvention employing polypeptone, therefore, has quite differentstructures and effects from conventional examples employing polypeptone.Moreover, there is no mention of the preference of the particularmonomer units which is the effect of the present invention, and noindication of the effect of the preference of the particular monomerunits having as substituents phenoxy, phenyl and cyclohexyl groups inthe composition of PHAs produced by the microorganisms, as in thepresent invention.

The PHAs, production methods, and microorganisms of the presentinvention will be explained in more detail below.

<Supplying Pathways of PHA Monomer Units>

At first will be detailed the “fatty acid synthesis pathway,” one ofpathways supplying mcl-3HA monomer units to be mixed into the PHA ofinterest.

In the case where saccharides such as glucose and the like aresubstrates, alkanoates necessary for cellular components arebiosynthesized form the “fatty acid synthesis pathway” in whichacetyl-CoA produced from saccharides through the “glycolytic pathway” isa starting substance. The fatty acid synthesis involves the de novosynthesis pathway and the carbon-chain elongation pathway, as explainedbelow.

1) De novo Synthesis Pathway

This pathway is catalyzed by two enzymes, acetyl-CoA carboxylase (EC6.4.1.2) and fatty acid synthase (E.C. 2.3.1.85). Acetyl-CoA carboxylaseis an enzyme interposing biotin, and ultimately catalyzing the followingreaction to produce malonyl-CoA from acetyl-CoA. This reaction is offollows:

acetyl-CoA+ATP+HCO₃ ⁻⇄malonyl-CoA+ADP+Pi

Also, fatty acid synthase is an enzyme catalyzing a cycles of reactionsof transfer—decarbonation—reduction—dehydration—reduction. The entirereactions are represented as follows:

acetyl-CoA+n malonyl-CoA+2n NADPH+2n H⁺→CH₃(CH₂)_(2n)COOH+n CO₂+2nNADP⁺+(n−1)CoA

Reaction products may be free acids, CoA-derivatives, orACP-derivatives, depending on the type of enzymes.

Now, acetyl-CoA and malonyl-CoA are represented by the followingchemical formulae (42) and (43), respectively.

In addition, Co-A stands for co-enzyme A, and is represented by thefollowing chemical formula (44).

Within this reaction pathway, the route described below gives“D-3-hydroxyacyl-ACP,” an intermediate to be the monomer substrate forthe PHA biosynthesis. Additionally, as shown in the following reactionformulae, this route extends finally to palmitate with repeated additionof two carbons. Therefore, as the monomer substrate for the PHAbiosynthesis are provided seven “D-3-hydroxyacyl-ACPs” having evennumbers of the carbons, from “D-3-hydroxybutyryl-ACP” to“D-3-hydroxypalmityl-ACP.”

2) Carbon-Chain Elongation Pathway

This pathway is broadly divided into two pathways: a pathway in whichmalonyl-CoA is added to acyl-ACP, which is finally converted to acyl-ACPhaving the carbon chain extended with two carbons (and CO₂) (referred toas Pathway A) and a pathway in which acetyl-CoA is added to acyl-CoA,which is finally converted to acyl-CoA having the carbon chain extendedwith two carbons (referred to as Pathway B). Each pathway will beexplained below.

Pathway A

 R—CO—ACP+malonyl-ACP→R—CO—CH₂—CO—ACP+CO₂R—CO—CH₂—CO—ACP→R—CHOH—CH₂—CO—ACP→R—CH═CH—CO—ACP→R—CH₂—CH₂—CO—ACP

Pathway B

R—CO—CoA+acetyl-CoA R—CO—CH₂—CO—CoA R—CO—CH₂—CO—CoAR—CHOH—CH₂—CO—CoA→R—CH═CH—CO—CoA→R—CH₂—CH₂—CO—CoA

In either Pathway A or B, it is thought that “D-3-hydroxyacyl-CoA” or“D-3-hydroxyacyl-ACP” is yielded as an intermediate, and“D-3-hydroxyacyl-CoA” is utilized as the monomer substrate for the PHAsynthesis as it is, while “D-3-hydroxyacyl-ACP” is utilized as themonomer substrate for the PHA synthesis after converting to“D-3-hydroxyacyl-CoA” by ACP—CoA transferase.

In the case where saccharides such as glucose and the like are used as asubstrate, it is thought that an mcl-3HA monomer unit is generatedwithin microbial cells via the “glycolytic pathway” and the “fatty acidsynthesis pathway” as described above. In the case where organic acidsinvolved in the TCA cycle are used as a substrate, acetyl-CoA is yieldeddirectly from pyruvic acid by pyruvate dehydrogenase. Organic acids inthe TCA cycle, for example, malic acid yields pyruvic acid by malatedehydrogenase, followed by acetyl-CoA by the above-mentioned reaction.Oxaloacetic acid yields phosphoenolpyruvic acid by phosphoenolpyruvatecarboxykinase, which, in turn, is catalyzed to produce pyruvic acid bypyruvate kinase, followed by acetyl-CoA by the above-mentioned reaction.It is thought that acetyl-CoA produced by these reactions goes throughthe “fatty acid synthesis pathway” to produce an mcl-3HA monomer unit.

In these cases, it is thought that mcl-alkanoates, for example,octanoate, nonanoate, and the like, or alkanoates having functionalgroups other than straight aliphatic alkyl added at the end, forexample, such as 5-phenylvalerate, 5-(4-fluorophenyl)valerate,6-phenylhexanoate, 4-phenoxybutyrate, and 4-cyclohexylbutyrate areconverted to their CoA-derivatives by CoA-ligases (EC 6.2.1.3, etc),followed by “D-3-hydroxyacyl-CoA” to be the monomer substrate for thePHA biosynthesis directly by a series of enzymes responsible for theβ-oxidation pathway.

In short, the mcl-3HA monomer units generated from saccharides ororganic acids involved in the TCA cycle is produced via quite a lot ofenzymatic reaction steps (i.e., indirectly), but the mcl-alkanoatesshould yield the mcl-3HA monomer units quite directly.

There will now be described the generation of acetyl-CoA responsible formicrobial growth. In the method in which mcl-alkanoate is co-existed inaddition to alkanoate for introducing the monomer unit of interest,these alkanoates go through the β-oxidation pathway to produceacetyl-CoA. Generally, mcl-alkanoates are believed to have a superiorsubstrate-affinity for a series of enzymes in the β-oxidation pathway,as compared with alkanoates having a bulky substituent (alkanoateshaving substituents such as phenyl, phenoxy, cyclohexyl group, or thelike), and thus acetyl-CoA is effectively produced by co-existence withmcl-alkanoates. For.this reason, it is advantageous to microbial growthutilizing acetyl-CoA as the energy and carbon source.

However, since mcl-alkanoates pathway are directly converted to themonomer unit for PHA via the β-oxidation, it is a significant problemthat the produced PHAs also contains the mcl-3HA monomer unit a lot, inaddition to -the monomer unit of interest.

To solve this problem, methods are desirable in which rather thanmcl-alkanoate, such substrates that can provide acetyl-CoA or the energyand carbon source effectively are selected and allowed to co-exist withthe alkanoate of interest. As mentioned above, acetyl-CoA can be themonomer unit of PHA by going into the fatty acid synthesis pathway, butthis process is an indirect process of many steps compared withmcl-alkanoate in β-oxidation. Therefore, it is possible to achieve aproduction method not to incorporate or decrease mcl-3HA in PHA byselecting culture conditions such as the substrate concentration togenerate acetyl-CoA.

Alternatively, there are commonly used production methods by whichculture is carried out at the first step only for the purpose ofmicrobial growth and at the second step is added to the medium only thealkanoate of interest as the carbon source. In this case, ATP isrequired by acyl-CoA ligase which is an initial enzyme of thebeta-oxidation pathway converting the alkanoate to acyl-CoA.Consequently, the inventors' investigation has provided the result thatthe production methods are-more effective by which substrates capable ofbeing utilized as the energy source by microorganisms are alsoco-existed at the second step, and accomplished the present invention.

As substrates which can effectively provide acyl-CoA or the energy andcarbon source in method according to the present invention, as long ascompounds can yield acyl-CoA or the energy and carbon source withoutgoing through the beta-oxidation pathway, for example, aldoses includingglyceraldehyde, erythrose, arabinose, xylose, glucose, galactose,mannose, and fructose, alditols such as glycerol, erythritol, andxylitol, aldonic acids such as gluconic acid, uronic acids such asglucuronic acid and galacturonic acid, saccharides such as disaccharidesincluding maltose, sucrose, and lactose, and in addition, organic acidsinvolved in the TCA cycle such as lactic, pyruvic, malic, citric,succinic, fumaric acids and their salts, and further, medium componentsderived from natural products such as polypeptone, beef extract, andcasamino acid, and the like, any compounds can be used, or selected asappropriate, based on usefulness as a substrate for the strainsemployed. Furthermore, if their combinations result in a small degree ofmixture with mcl-3HA, two or more compounds also can be selected foruse.

<Microorganisms>

As mentioned in Background of the Invention, there are reports ofmicroorganisms that produce and accumulate within the cell PHAcontaining the monomer unit of, for example,3-hydroxy-4-phenoxybutyrate, 3-hydroxy-5-fluorophenoxyvalerate,3-hydroxy-6-cyanophenoxyhexanoate, 3-hydroxy-6-nitrophenoxyhexanoate,3-hydroxy-7-fluorophenoxyheptanoate, such as Pseudomonas oleovorance andPseudomonas putida as described in Macromolecules, 29, 3432-3435 (1996),Can. J. Microbiol., 41, 32-43 (1995), Japanese Patent No. 2989175, andothers. However, there are no report of microorganisms that produce andaccumulate within the cell PHA containing the monomer unit of3-hydroxy-4-phenoxybutyrates having substituents such as fluorine,cyano, nitro groups. Macromolecules, 32, 2889-2895 (1999) reported thatPseudomonas oleovorance produces PHA containing the monomer units of3-hydroxy-5-(2,4-dinitrophenyl)valerate and3-hydroxy-5-(4-nitrophenyl)valerate by culturing it in a mediumcontaining 5-(2,4-dinitrophenyl)valerate and nonanoate as substrates.However, there are no reports on microorganisms that produce andaccumulate within the cell PHA containing as the monomer unit3-hydroxy-phenylalkanoates having a substituent such as fluorine,trifluoromethyl group. Therefore, the present invention has beenachieved by screening microorganisms capable of incorporating these newmonomer units into PHA.

Novel microorganisms of the present invention have a previously unknowncapability of producing and accumulating within the cell PHA containinga new monomer unit derived from an alkanoate using the alkanoate as asubstrate. Microorganisms displaying such a novel enzymatic reactionhave been found by the inventors by screening. The novel microorganismsof the present invention are Pseudomonas cichorii strain YN2 (FERMBP-7375), Pseudomonas cichorii strain H45 (FERM BP-7374), Pseudomonasputida strain P91 (FERM BP-7373), and Pseudomonas jessenii strain P161(FERM BP-7376). Other than these microorganisms, microorganisms to beutilized in the production method of PHA according to the presentinvention can be obtained by culturing a bacterial strain, for example,of genus Pseudomonas, employing the alkanoates as the substrate, forexample.

There will be given details concerning strains YN2, H45, P91, and P161.

<Bacteriological Properties of Strain YN2>

(1) Morphological Properties

Shape and size of cells: rod, 0.8 μm×1.5 to 2.0 μm

Polymorphism of cells: negative

Mobility: motile

Sporulation: negative

Gram staining: negative

Colony shape: circular; entire, smooth margin; low convex; smoothsurface; glossy; translucent

(2) Physiological Properties

Catalase: positive

Oxidase: positive

O/F test: oxidative (non-fermentative)

Nitrate reduction: negative

Indole production: positive

Acid production from glucose: negative

Arginine dihydrolase: negative

Urease: negative

Esculin hydrolysis: negative

Gelatin hydrolysis: negative

β-Galactosidase: negative

Fluorescent pigment production on King's B agar: positive

Growth under 4% NaCl: positive (weak growth)

Poly-β-hydroxybutyrate accumulation: negative (*)

Tween 80 hydrolysis: positive

(*) Colonies cultured on nutrient agar were stained with Sudan Black fordetermination.

(3) Substrate Assimilation

Glucose: positive

L-Arabinose: positive

D-Mannose: negative

D-Mannitol: negative

N-Acetyl-D-glucosamine: negative

Maltose: negative

Potassium gluconate: positive

n-Caprate: positive

Adipate: negative

dl-Malate: positive

Sodium citrate: positive

Phenyl acetate: positive

<Bacteriological Properties of Strain H45>

(1) Morphological Properties

Shape and size of cells: rod, 0.8 μm×1.0 to 1.2 μm

Polymorphism of cells: negative

Mobility: motile

Sporulation: negative

Gram staining: negative

Colony shape: circular; entire, smooth margin; low convex; smoothsurface; glossy; cream-colored

(2) Physiological Properties

Catalase: positive

Oxidase: positive

O/F test: oxidative

Nitrate reduction: negative

Indole production: negative

Acid production from glucose: negative

Arginine dihydrolase: negative

Urease: negative

Esculin hydrolysis: negative

Gelatin hydrolysis: negative

β-Galactosidase: negative

Fluorescent pigment production on the King's B agar: positive

Growth under 4% NaCl: negative

Poly-β-hydroxybutyrate accumulation: negative

(3) Ability to Assimilate Substrates

Glucose: positive

L-Arabinose: negative

D-Mannose: positive

D-Mannitol: positive

N-Acetyl-D-glucosamine: positive

Maltose: negative

Potassium gluconate: positive

n-Caprate: positive

Adipate: negative

dl-Malate: positive

Sodium citrate: positive

Phenyl acetate: positive

<Bacteriological Properties of Strain P91>

(1) Morphological Properties

Shape and size of cells: rod, 0.6 μm×1.5 μm

Polymorphism of cells: negative

Mobility: motile

Sporulation: negative

Gram staining: negative

Colony shape: circle; entire, smooth margin; low convex; smooth surface;glossy; cream-colored

(2) Physiological Properties

Catalase: positive

Oxidase: positive

O/F test: oxidative

Nitrate reduction: negative

Indole production: negative

Acid production from glucose: negative

Arginine dihydrolase: positive

Urease: negative

Esculin hydrolysis: negative

Gelatin hydrolysis: negative

β-Galactosidase: negative

Fluorescent pigment production on the King's B agar: positive

(3) Substrate Assimilation

Glucose: positive

L-Arabinose: negative

D-Mannose: negative

D-Mannitol: negative

N-Acetyl-D-glucosamine: negative

Maltose: negative

Potassium gluconate: positive

n-Caprate: positive

Adipate: negative

dl-Malate: positive

Sodium citrate: positive

Phenyl acetate: positive

<Bacteriological Properties of the Strain P161>

(1) Morphological Properties

Shape and size of cells: spheres, φ0.6 μm rods, 0.6 μm×1.5 to 2.0 μm

Polymorphism of cells: elongated form

Mobility: motile

Sporulation: negative

Gram staining: negative

Colony shape: circle; entire, smooth margin; low convex; smooth surface;pale yellow

(2) Physiological Properties

Catalase: positive

Oxidase: positive

O/F test: oxidative

Nitrate reduction: positive

Indole production: negative

Acid production from glucose: negative

Arginine dihydrolase: positive

Urease: negative

Esculin hydrolysis: negative

Gelatin hydrolysis: negative

β-Galactosidase: negative

Fluorescent pigment production on the King's B agar: positive

(3) Substrate Assimilation

Glucose: positive

L-Arabinose: positive

D-Mannose: positive

D-Mannitol: positive

N-Acetyl-D-glucosamine: positive

Maltose: negative

Potassium gluconate: positive

n-Caprate: positive

Adipate: negative

dl-Malate: positive

Sodium citrate: positive

Phenyl acetate: positive

Based on these bacteriological properties, and referring to Bergey'sManual of Systematic Bacteriology, vol. 1 (1984) and Bergey's Manual ofDeterminative Bacteriology, 9th ed. (1994), strains YN2 and H4.5 wererevealed to belong to Pseudomonas cichorii, and strain P91 was revealedto belong to Pseudomonas putida. Accordingly, these strains weredesignated Pseudomonas cichorii strain YN2, Pseudomonas cichorii strainH45, and Pseudomonas putida strain P91.

Strain P161, on the other hand, was revealed to belong to the genusPseudomonas (Pseudomonas sp.), but its bacteriological properties couldnot identify its taxonomic position. Then, to do classification based ongenetic properties, the DNA sequence of 16S rRNA coding region of strainP161 (SEQ ID NO: 1) has been determined to examine the homology with theDNA sequences of 16S RNA coding region of known microorganisms of genusPseudomonas. The results have shown that there is a remarkably highhomology of the DNA sequences between strain P161 and Pseudomonasjessenii. Furthermore, bacteriological properties of Pseudomonasjessenii described in System. Appl. Microbiol., 20, 137-149 (1997) andSystem. Appl. Microbiol., 22, 45-58 (1999) were found to have a highsimilarity to those of strain P161. From these results, strain P161 wasdesignated Pseudomonas jessenii strain P161, since it was concluded thatit is appropriate that strain P161 should be attributed to belong toPseudomonas jessenii.

Strains YN2, H45, P91, and P161 have been deposited at NationalInstitute of Bioscience and Human-Technology (Patent MicroorganismDepository Center), Agency of Industry Science and Technology, Ministryof International Trade and Industry, under the deposition numbers “FERMBP-7375,” “FERM BP-7374, ” “FERM BP-7373,” and “FERM BP-7376, ”respectively.

<Culture: General>

The PHAs of interest can be produced by culturing these microorganismsin a medium containing alkanoate for introducing the desired monomerunit and growth substrates according to the present invention. ThesePHAs -are generally of only the R-form, and are isotactic polymers.

For usual culture of microorganisms to be employed in the productionmethod of PHA according to the present invention, for example, forpreparation of cell stocks, for maintaining of the number and activitiesof the cells, any type of media can be used, such as common naturalmedia and synthetic media supplemented with nutrients, unless they haveadverse effects on the growth or existence of the microorganisms.Culture conditions such as temperature, aeration, stirring, and the likeare selected as appropriate, depending on the microorganism employed.

In the case where microorganisms are used to produce and accumulate PHA,inorganic media and others containing alkanoate for introducing thedesired monomer unit can be employed as a medium for the PHA production.

For inorganic media employed in the above-mentioned culture method, anymedia can be used, as long as they contain components allowingmicroorganisms to grow, such as phosphorus sources (for example,phosphates), nitrogen sources (for example, ammonium salts, nitrates),and the like. Such inorganic media may include, for example, MSB medium,E medium (J. Biol. Chem. 218, 97-106 (1956)), M9 medium, and others.

The composition of M9 medium employed in Examples of the presentinvention is as follows:

Na₂HPO₄: 6.2 g KH₂PO₄: 3.0 g NaCl: 0.5 g NH₄Cl: 1.0 g

(per litter of medium, pH 7.0)

Culture conditions may include, for example, shaking culture andstirring culture under aerobic conditions at 15 to 40° C., andpreferably 20 to 35° C.

The culture steps can utilize any processes employed for usual cultureof microorganisms, such as batch, flow batch, semi-continuous,continuous, and reactor-type cultures, and may take multi-step processesconnecting plural steps of these processes.

For respective growth substrates, specific culture steps will bedescribed as follows:

<Culture: mcl-Alkanoates>

As a method of, for example, two-step culture, there is a method bywhich the first-step culture is carried out in an inorganic medium orthe like containing a first alkanoate having 6 to 12 carbon atoms, suchas octanoate and nonanoate, as the growth substrate at an amount of theorder of 0.1% by weight to 0.2% by weight and a second alkanoate forintroducing the desired monomer unit at an amount of the order of 0.01%by weight to 0.5% by weight until the time of the late logarithmicgrowth phase to the stationary phase, and at the second step, cellsafter the first-step culture is completed are collected bycentrifugation or the like, followed by further culturing them in aninorganic medium containing the second alkanoate at an amount of theorder of 0.01% by weight to 0.5% by weight and no nitrogen sources, andafter the culture is completed, the cells are harvested to extract thedesired PHA.

Alternatively, there is another method by which culture is carried outby supplying a first alkanoate having 6 to 12 carbon atoms, such asoctanoate and nonanoate, at an amount of the order of 0.1% by weight to0.2% by weight and a second alkanoate for introducing the desiredmonomer unit at an amount of the order of 0.01% by weight to 0.5% byweight, and cells are harvested at the time of the late logarithmicgrowth phase to the stationary phase to extract the desired PHA.

In the methods in which mcl-alkanoate as the growth substrate is addedto the medium, the obtained PHAs are ones in which is mixed a largeamount of the monomer unit derived from the mcl-alkanoate added as thegrowth substrate. Such PHAs are generally of only the R form, and areisotactic polymers.

<Culture: Saccharides>

As a method, for example, a two-step culture, there is a method by whichthe first-step culture is carried out in an inorganic medium or the likecontaining saccharide(s) (for example, glucose, mannose, fructose, etc.)as the growth substrate at an amount of the order of 0.1% by weight to2.0% by weight and alkanoate for introducing the desired monomer unit atan amount of the order of 0.01% by weight to 0.5% by weight until thetime of the late logarithmic growth phase to the stationary phase, andat the second step, cells after the first-step culture is completed arecollected by centrifugation or the like, followed by further culturingthem in an inorganic medium containing saccharide(s) (for example,glucose, mannose, fructose, etc.) as the growth substrate at an amountof the order of 0.1% by weight to 2.0% by weight, the alkanoate at anamount of the order of 0.01% by weight to 0.5% by weight, and nonitrogen sources, and after the culture is completed, the cells areharvested to extract the desired PHA.

Alternatively, there is another method by which culture is carried outby supplying saccharide(s) (for example, glucose, mannose, fructose,etc.) as the growth substrate at amounts of the order of 0.1% by weightto 2.0% by weight and alkanoate for introducing the desired monomer unitat an amount of the order of 0.01% by weight to 0.5% by weight, andcells are harvested at the time of the late logarithmic growth phase tothe stationary phase to extract the desired PHA.

In these cases, the concentration of the saccharides (for example,glucose, mannose, fructose, etc.) to be added to the medium is selectedas appropriate, depending on the type of alkanoate for introducing thedesired monomer unit, the genus and species of the microorganism, thecell density, or the culture process, although addition can be selectedsuch that the content in the medium is usually in the order of 0.1% byweight to 2.0% by weight. On the other hand, the concentration of thealkanoate to be raw material is also selected as appropriate, dependingon the genus and species of the microorganism, the cell density, or theculture process, although addition can be selected such that the contentin the medium is usually in the order of 0.01% by weight to 0.5% byweight. Thus, by culturing the microorganism in a medium containingsaccharide(s) (for example, glucose, mannose, fructose, etc.) and thealkanoate, the desired PHAs can be produced and accumulated in which themonomer unit other than the intended one is incorporated at a smallamount or not at all. These PHAs are generally of only the R form, andare isotactic polymers.

<Culture: Polypeptone>

As a method of, for example, a two-step culture, there is a method bywhich the first-step culture is carried out in an inorganic medium orthe like containing polypeptone as the growth substrate at an amount ofthe order of 0.1% by weight to 2.0% by weight and alkanoate forintroducing the desired monomer unit at an amount of the order of 0.01%by weight to 0.5% by weight until the time of the late logarithmicgrowth phase to the stationary phase, and at the second step, cellsafter the first-step culture is completed are collected bycentrifugation or the like, followed by further culturing them in aninorganic medium containing the alkanoate at an amount of the order of0.01% by weight to 0.5% by weight, and no nitrogen sources, and afterthe culture is completed, the cells are harvested to extract the desiredPHA.

Alternatively, there is another method by which culture is carried outby supplying polypeptone at an amount of the order of 0.1% by weight to2.0% by weight and alkanoate for introducing the desired monomer unit atan amount of the order of 0.01% by weight to 0.5% by weight, and cellsare harvested at the time of the late logarithmic growth phase to thestationary phase to extract the desired PHA.

In these cases, the concentration of polypeptone to be added to themedium is selected as appropriate, depending on the type of alkanoatefor introducing the desired monomer unit, the genus and species of themicroorganism, the cell density, or the culture process, althoughaddition can be selected such that the content in the medium is usuallyin the order of 0.1% by weight to 2.0% by weight. For polypeptone, it isalso possible to use, as appropriate, any commercial availablepolypeptone that is commonly employed for culturing microorganisms andthe like. On the other hand, the concentration of the alkanoate to beraw material is also selected as appropriate, depending on the genus andspecies of the microorganism, the cell density, or the culture process,although addition may be selected such that the content in the medium isusually in the order of 0.01% by weight to 0.5% by weight. Thus, byculturing the microorganism in a medium containing polypeptone and thealkanoate, the desired PHAs can be produced and accumulated in which themonomer unit other than the intended one is incorporated at a smallamount or not at all. These PHAs are generally of only the R form, andare isotactic polymers.

<Culture: Organic Acids of TCA Cycle>

As a method of, for example, a two-step. culture, there is a method bywhich the first-step culture is carried out in an inorganic medium orthe like containing organic acid(s) involved in the TCA cycle (forexample, lactic, pyruvic, citric, succinic, fumaric, malic acids and thelike, and salts thereof) as the growth substrate at an amount of theorder of 0.1% by weight to 2.0% by weight and alkanoate for introducingthe desired monomer unit at an amount of the order of 0.01% by weight to0.5% by weight until the time of the late logarithmic growth phase tothe stationary phase, and at the second step, cells after the first-stepculture is completed are collected by centrifugation or the like,followed by further culturing them in an inorganic medium containingorganic acid(s) of the TCA cycle (for example, lactic, pyruvic, citric,succinic, fumaric, malic acids and the like, and salts thereof) as thegrowth substrate at an amount of the order of 0.1% by weight to 2.0% byweight, the alkanoate at an amount of the order of 0.01% by weight to0.5% by weight, and no nitrogen sources, and after the culture iscompleted, the cells are harvested to extract the desired PHA.

Alternatively, there is another method by which culture is carried outby supplying organic acid(s) of the TCA cycle (for example, lactic,pyruvic, citric, succinic, fumaric, malic acids and the like, and saltsthereof) at an amount of the order of 0.1% by weight to 2.0% by weightand alkanoate for introducing the desired monomer unit at an amount ofthe order of 0.01% by weight to 0.5% by weight, and cells are harvestedat the time of the late logarithmic growth phase to the stationary phaseto extract the desired PHA.

In these cases, the concentration of organic acids of the TCA cycle (forexample, lactic, pyruvic, citric, succinic, fumaric, malic acids and thelike, and salts thereof) to be added to the medium is selected asappropriate, depending on the type of alkanoate for introducing thedesired monomer unit, the genus and species of the microorganism, thecell density, or the culture process, although addition can be selectedsuch that the content in the medium is usually in the order of 0.1% byweight to 2.0% by weight. On the other hand, the concentration of thealkanoate to be raw material is also selected as appropriate, dependingon the genus and species of the microorganism, the cell density, or theculture process, although addition can be selected such that the contentin the medium is usually in the order of 0.01% by weight to 0.5% byweight. Thus, by culturing the microorganism in a medium containingorganic acid(s) of the TCA cycle (for example, lactic, pyruvic, citric,succinic, fumaric, malic acids and the like, and salts thereof) and thealkanoate, the desired PHAs can be produced and accumulated in which themonomer unit other than the intended one is incorporated at a smallamount or not at all. These PHAs are generally of only the R form, andare isotactic polymers.

<Culture: Polypeptone+Pyruvic Acid and Salts Thereof>

As a method of, for example, a two-step culture, there is a method bywhich the first-step culture is carried out in an inorganic medium orthe like containing polypeptone as the growth substrate at an amount ofthe order of. 0.1% by weight to 2.0% by weight and alkanoate forintroducing the desired monomer unit at an amount of the order of 0.01%by weight to 0.5% by weight until the time of the late logarithmicgrowth phase to the stationary phase, and at the second step, cellsafter the first-step culture is completed are collected bycentrifugation or the like, followed by further culturing them in aninorganic medium containing pyruvic acid or salt thereof as the growthsubstrate at an amount of the order of 0.1% by weight to 2.0% by weight,the alkanoate at an amount of the order of 0.01% by weight to 0.5% byweight, and no nitrogen sources, and after the culture is completed, thecells are harvested to extract the desired PHA.

In these cases, the concentration of polypeptone and pyruvic acid orsalt thereof to be added to the medium is selected as appropriate,depending on the type of alkanoate for introducing the desired monomerunit, the genus and species of the microorganism, the cell density, orthe culture process, although addition can be selected such that thecontent in the medium is usually in the order of 0.1% by weight to 2.0%by weight in each case. On the other hand, the concentration of thealkanoate to be raw material is also selected as appropriate, dependingon the genus and species of the microorganism, the cell density, or theculture process, although addition can be selected such that the contentin the medium is usually in the order of 0.01% by weight to 0.5% byweight. Thus, by culturing the microorganism in two steps utilizing amedium containing polypeptone and the alkanoate and a medium containingpyruvic acid or salt thereof and the alkanoate, the desired PHAs can beproduced and accumulated in which the monomer unit other than theintended one is incorporated at a small amount or not at all. These PHAsare generally of only the R form, and are isotactic polymers.

<PHA Recovery>

For PHA recovery from cells in the method according to the presentinvention, usually-operating extraction with organic solvents such aschloroform is most convenient, but in the circumstances where it isdifficult to use organic solvents, it is also possible to utilizemethods of collecting PHA by removing cellular components other than PHAby means of treatment with detergents such as SDS and the like,treatment with enzymes such as lysozyme and the like, treatment withchemicals such as EDTA, sodium hypochlorite, ammonia, and the like.

<Molecular Weight>

The PHAs according to the present invention can be obtained by utilizingthe above-mentioned methods. It is desirable that the PHAs have a numberaverage molecular weight of more than at least 10,000 or so, in order toallow stable physical properties as polymer, for example, such as glasstransition temperature, softening point, melting point, crystallinity,orientation defined by the monomer unit of which the polymer iscomposed, to be fixed. The PHAs according to the present invention havea number average molecular weight of about 20,000 or higher, andtherefore can be sufficiently expected to display stable physicalproperties as polymer. From the viewpoint of convenience of treatmentssuch as dissolving processes, the PHAs preferably have a number averagemolecular weight of up to 200,000 or so, and more preferably not morethan 100,000. As mentioned above, these PHAs are generally composed ofonly the R form, and are isotactic polymers.

Culturing of the microorganisms, production and accumulation of PHAswithin cells by the microorganisms, and recovery of PHAs from the cellsare not limited to the methods described above. For example, in additionto four strains described above, microorganisms to be employed in themethod of producing PHAs according to the present invention can utilizemicroorganisms having similar production capabilities of producing PHAsaccording to the present invention as those of these four strains.

It is likely that these PHAs are useful, for example, for device andmedical materials and others, as well as applications in which commonplastics are used. Those having fluorine atom(s), trifluoromethyl group,and the like introduced as a substituent group, in particular, areexpected to have a superior biocompatibility, and therefore applicationsto medical uses. Furthermore, they are predicted to have a water-repellent effect due to containing fluorine atom(s), trifluoromethylgroup, and the like, and thus applications to water-repellent treatmentsin various fields are also possible. Specifically, applications totemporary water-repellent treatments also can be contemplated utilizingbiodegradability resulting from aliphatic polyesters.

EXAMPLES Example 1

The substrate FPVA was first synthesized by the Grignard reactionaccording to the method described in “Macromolecules, 29, 1762-1766(1996) and 27, 45-49 (1994)”. 5-bromovaleric acid was dissolved inanhydrous tetrahydrofuran (THF), and 3 mol/L of a methyl magnesiumchloride THF solution was added dropwise at −20° C. in an argonatmosphere. After stirring for about 15 min, a THF solution of1-bromo-4-fluorobenzene and magnesium was further dropped and a THFsolution of 0.1 mol/L Li₂CuCl₄ was added (temperature was maintained at−20° C.). The reaction solution was resumed to room temperature andfurther stirred overnight. Then the solution was poured into a 20%sulfuric acid solution cooled on ice, and stirred. The aqueous layer wasrecovered, saturated with salt, and extracted with ether. After theextract was further extracted with 100 mL of deionized water, to which50 g of potassium hydroxide was added, the extract was acidified with a20% sulfuric acid solution and the precipitate was recovered.

The precipitate was analyzed with nuclear magnetic resonance equipment(FT-NMR: Bruker DPX400) under the following conditions: nuclide: ¹H and¹³C; solvent: heavy chloroform (containing TMS). The results are shownin FIG. 1 and Table 3.

Example 2

Cells of strain H45 was inoculated in 200 mL of M9 medium containingnonanoic acid 0.1% and FPVA 0.1%, and shake-cultured at 30° C. at 125strokes/min. After 24 hours, the cells were collected by centrifugation,resuspended in 200 mL of M9 medium containing FPVA 0.2% but not anitrogen source (NH₄Cl), and further shake-cultured at 30° C. at 125strokes/min. After 24 hours, the cells were collected by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform and stirredat 60° C. for 20 hours to extract PHA. After the extract was filteredwith a membrane filter of 0.45 μm pore size, the filtrate wasconcentrated with a rotary evaporator and the concentrate wasreprecipitated in cold methanol. Only the precipitate was then recoveredand vacuum-dried to obtain PHA. After the PHA obtained was subjected tomethanolysis according to the conventional method, it was analyzed witha gas chromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EImethod) and the methyl esters of the PHA monomer unit were identified.The results are shown in Table 4.

Example 3

Cells of strain YN2 was inoculated in 200 mL of M9 medium containingnonanoic acid 0.1% and FPVA 0.1%, and shake-cultured at 30° C. at 125strokes/min. After 24 hours, the cells were collected by centrifugation,resuspended in 200 mL of M9 medium containing FVPA 0.2% but not anitrogen source (NH₄Cl), and further shaken-cultured at 30° C. at 125strokes/min. After 24 hours, the cells were collected by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform, stirred at60° C. for 20 hours to extract PHA. After the extract was filtered witha membrane filter of 0.45 μm in diameter, the filtrate was concentratedwith a rotary evaporator and the concentrate was reprecipitated in coldmethanol. The precipitate was then recovered and vacuum-dried to obtainPHA. After the PHA obtained was subjected to methanolysis according tothe conventional method, it was analyzed with a gas chromatograph-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) and the methyl estersof the PHA monomer unit were identified. The results are shown in Table5.

Example 4

Cells of strain P91 were inoculated in 200 mL of M9 medium containingnonanoic acid 0.1% and shaken-cultured at 30° C. at 125 strokes/min.After 24 hours, the cells were collected by centrifugation, resuspendedin 200 mL of M9 medium containing nonanoic acid 0.1% and FPVA 0.1% butnot a nitrogen source (NH₄Cl), and further shaken-cultured at 30° C. at125 strokes/min. After 24 hours, the cells were collected bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform and stirredat 60° C. for 20 hours to extract PHA. After the extract was filteredwith a membrane filter of 0.45 μm in diameter, the filtrate wasconcentrated with a rotary evaporator and the concentrate wasreprecipitated in cold methanol. Only the precipitate was then recoveredand vacuum-dried to obtain PHA. After the PHA obtained was subjected tomethanolysis according to the conventional method, it was analyzed witha gas chromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EImethod) and the methyl esters of the PHA monomer unit were identified.The results are shown in Table 6.

Example 5

Cells of strain P161 were inoculated in 200 mL of M9 medium containingnonanoic acid 0.1% and shaken-cultured at 30° C. at 125 strokes/min.After 24 hours, the cells were collected by centrifugation, resuspendedin 200 mL of M9 medium containing nonanoic acid 0.1% and FPVA 0. 1%butnot a nitrogen source (NH₄C1), and further shaken-cultured at 30° C. at125 strokes/min. After 24 hours, the cells were collected bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform and stirredat 60° C. for 20 hours to extract PHA. After the extract was filteredwith a membrane filter of 0.45 μm in diameter, the filtrate wasconcentrated with a rotary evaporator and the concentrate wasreprecipitated in cold methanol. Only the precipitate was then recoveredand vacuum-dried to obtain PHA. After the PHA obtained was subjected tomethanolysis according to the conventional method, it was analyzed witha gas chromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EImethod) and the methyl esters of the PHA monomer unit were identified.The results are shown in Table 7.

Example 6

After 100 mg of PHA derived from strain H45 was dissolved in 1 mL ofchloroform, n-hexane was added until it was clouded. This wascentrifuged to recover and vacuum-dried the precipitate. This was againdissolved in 1 mL of chloroform, n-hexane was added, and the procedureof recovering the precipitate was repeated three times.

After the precipitate obtained was subjected to methanolysis accordingto the conventional method, it was analyzed with a gaschromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) andthe methyl esters of the PHA monomer unit were identified. As a result,the precipitate was found to be PHA whose monomer unit consisted of3HFPV monomer alone as shown in Table 8.

In addition, nuclear magnetic resonance equipment (FT-NMR: BrukerDPX400) was used for analysis under the following condition: nuclide: ¹Hand ¹³C; solvent: heavy chloroform (containing TMS). The results areshown in FIG. 2, Table 9, FIG. 3 and Table 10.

Example 7

(Synthesis of FPxVA)

After 240 mL of dehydrated acetone was put into a three-neckedround-bottom flask, sodium iodide (0.06 mol), potassium carbonate (0.11mol) and 4-fluorophenol (0.07 mol) were added and thoroughly stirred.5-bromoethylvalerate (0.06 mol) was dropped into the solution in anitrogen atmosphere, refluxed at 60±5° C. and allowed to react for 24hours. After reaction, the reaction solution was concentrated to drynesswith an evaporator and redissolved in methylene chloride. Water wasadded and the solution was separated. The organic layer was dehydratedwith anhydrous magnesium sulfate and concentrated to dryness with anevaporator.

Hot methanol was added to the reactant, dissolved, slowly cooled andreprecipitated to obtain 5-(4-fluorophenoxy)ethylvalerate. At this time,the yield from 5-bromoethylvalerate was 68 mol %.

The reactant (ester) obtained was dissolved in ethanol-water (9.1 (v/v))so as to be 5 weight %. Ten-fold molar quantity of potassium hydroxidewas added and allowed to react at 0 to 4° C. for 4 hours to hydrolyzethe ester.

The reaction solution was poured into 10 volumes of a 0.1 mol/Lhydrochloric acid solution and the precipitate was recovered byfiltration. The precipitate (reactant) recovered was vacuum-dried atroom temperature for 36 hours. The dried substance obtained wasdissolved in a small quantity of hot ethanol, and the solution wasgradually cooled, reprecipitated, vacuum-dried at room temperature for24 hours to obtain the target compound 5-(4-fluorophenoxy)valeric acid.The yield of this compound from 5-bromoethyl valerate was 49 mol %.

The compound obtained was analyzed by NMR under the followingconditions:

<Equipment>

FT-NMR: Bruker DPX400

¹H resonance frequency: 400 MHZ

<Measurement conditions>

nuclide: ¹H

solvent: CDCl₃

reference: capillary-contained TMS/CDCI₃

temperature: room temperature

The spectral chart is shown in FIG. 4 and the results of identificationare shown in Table 11.

The above results confirmed that the desired FPxVA was certainlysynthesized.

Example 8

(Production of PHA by strain P91)

Cells of strain P91 were inoculated in 200 mL of M9 medium containingnonanoic acid 0.1 weight % and FPxVA 0.1 weight %, and shaken-culturedat 30° C. at 125 strokes/min. After 24 hours, the cells were collectedby centrifugation, resuspended in 200 mL of M9 medium containingnonanoic acid 0.1 weight % and FPxVA 0.1 weight % but not a nitrogensource (NH₄Cl), and further shaken-cultured at 30° C. at 125strokes/min. After 24 hours, the cells were collected by centrifugation,washed once with cold methanol and lyophilized.

After the lyophilized pellet was weighed, it was suspended in 100 mL ofchloroform and stirred at 60° C. for 20 hours to extract PHA. After theextract was filtered with a membrane filter of 0.45 μm in pore size, thefiltrate was concentrated with a rotary evaporator and the concentratewas reprecipitated in cold methanol. Only the precipitate was thenrecovered and vacuum-dried to obtain and weigh PHA. The yields are shownin Table 12.

The PHA obtained was measured for the molecular weight by gel permeationchromatography (GPC; Toso HCL-8020, column: Polymer Laboratory PLgelMIXED-C (5 μm), solvent: chloroform, converted on a polystyrene basis).The molecular weight is shown in Table 13.

In addition, after the PHA obtained was subjected to methanolysisaccording to the conventional method, it was analyzed by GC-MS and themethyl esters of the PHA monomer unit were identified. The TIC and themass spectrum of each peak are shown in FIG. 5 to FIGS. 7A and 7B. Peak(1) was shown to represent 3-hydroxymethylheptanoate, Peak (2)3-hydroxymethyloctanoate, Peak (3) 3-hydroxymethylnonanoate, and Peak(4) 3 hydroxy-4-(4-fluorophenoxy)methylvalerate.

The above results indicated that the polymer obtained was PHA containingthe units of 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid,3-hydroxynonanoic acid and 3-hydroxy-4-(4-fluorophenoxy)valeric acid.

Example 9

(Production of PHA by strain YN2 (1))

Except that strain P91 was replaced by strain YN2, the same proceduresas described in Example 8 were used to produce PHA, and each analysiswas performed. The yields are shown in Table 12, the molecular weight inTable 13, and the TIC of GC-MS in FIG. 8. Peak (1) was shown torepresent 3-hydroxymethylheptanoate, Peak (2) 3-hydroxymethyloctanoate,Peak (3) 3-hydroxymethylnonanoate, and Peak (4)3-hydroxy-4-(4-fluorophenoxy)methylvalerate.

The above results indicated that the polymer obtained was PHA containingthe units of 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid,3-hydroxynonanoic acid and 3-hydroxy-4-(4-fluorophenoxy)valeric acid.

Example 10

(Production of PHA by strain P161)

Except that strain P91 was replaced by strain P161, the same proceduresas described in Example 8 were used to produce PHA, and each analysiswas performed. The yields are shown in Table 12, the molecular weight inTable 13, and the TIC of GC-MS in FIG. 9. Peak (1) was shown torepresent 3-hydroxymethylheptanoate, Peak (2) 3-hydroxymethyloctanoate,Peak (3) 3-hydroxymethylnonanoate, and Peak (4) 3hydroxy-4-(4-fluorophenoxy)methylvalerate.

The above results indicated that the polymer obtained was PHA containingthe units of 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid,3-hydroxynonanoic acid and 3-hydroxy-4-(4-fluorophenoxy)valeric acid.

Example 11

(Production of PHA by strain H45)

Except that strain P91 was replaced by H45 strain, the same proceduresas described in Example 8 were used to produce PHA, and each analysiswas performed. The yields are shown in Table 12, the molecular weight inTable 13, and the TIC of GC-MS in FIG. 10. Peak (1) was shown torepresent 3-hydroxymethylheptanoate, Peak (2) 3-hydroxymethyloctanoate,Peak (3) 3-hydroxymethylnonanoate, and Peak (4)3-hydroxy-4-(4-fluorophenoxy)methylvalerate.

The above results indicated that the polymer obtained was PHA containingthe units of 3-hydroxyheptanoic-acid, 3-hydroxyoctanoic acid,3-hydroxynonanoic acid and 3-hydroxy-4-(4-fluorophenoxy)valeric acid.

Example 12

(Production of PHA by strain YN2 (2))

Cells of strain YN2 were inoculated in 200 mL of M9 medium containinghexanoic acid 0.1 weight % and FPxVA 0.1 weight %, and shaken-culturedat 30° C. at 125 strokes/min. After 72 hours, the cells were collectedby centrifugation and resuspended in 200 mL of M9 medium containinghexanoic acid 0.1 weight % and FPxVA 0.1 weight % but not a nitrogensource (NH₄Cl), and further shaken-cultured at 30° C. at 125strokes/min. After 30 hours, the cells were collected by centrifugation,washed once with cold methanol and lyophilized.

PHA was extracted with the same procedures as described in Example 8,and each analysis was performed. The yields are shown in Table 12 andthe molecular weight is shown in Table 13. The results of GC-MS analysisrevealed that the PHA obtained by this method had the followingcomposition:

3-hydroxybutyric acid: 8.1%

3-hydroxyhexanoic acid: 51.2%

3-hydroxyoctanoic acid: 1.3%

3-hydroxydecanoic acid: 7.0%

3-hydoxydodecanoic acid: 10.6%

unidentified substances: 9.9%

3 hydroxy-4-(4-fluorophenoxy)valeric acid: 11.9%

The above results indicated that the polymer obtained was PHA containingthe units of 3-hydroxy-4-(4-fluorophenoxy)valeric acid.

Example 13

(Synthesis of TFMPVA)

The substrate TFMPVA was first synthesized by the Grignard reactionaccording to the method described in “Macromolecules, 29, 1762-1766(1996) and 27, 45-49 (1994).” 5-bromovaleric acid was dissolved inanhydrous tetrahydrofuran (THF) and 3 mol/L of a methyl magnesiumchloride THF solution was added dropwise at −20° C. in an argonatmosphere. After stirring for about 15 min, a THF solution of1-bromo-4-trifluorobenzene and magnesium was further dropped and a THFsolution of 0.1 mol/L Li₂CuCl₂ was added (temperature was maintained at−20° C.). The reaction solution was restored to room temperature andfurther stirred overnight. Then the solution was poured into a 20%sulfuric acid solution cooled on ice, and stirred. The aqueous layer wasrecovered, saturated with salt, and extracted with ether. After theextract was further extracted with 100 mL of deionized. water, to which50 g of potassium hydroxide was added, the extract was acidified with a20% sulfuric acid solution and the precipitate was recovered.

The compound recovered was methylesterified by the conventional methodand analyzed with a gas chromatograph-mass spectrometer (GC-MS, ShimadzuGC-MS QP-5050, column: DB-WAXETR (30 m×0.32 mm×0.5 μm) (manufactured byJ&W Inc.) The TIC (total ion chromatogram) and mass spectrum are shownin FIGS. 11A and 11B. These results revealed that the target TFMPVA wassynthesized.

Example 14

(Production of polymer by strain H45)

Cells of strain H45 were inoculated in 200 mL of M9 medium containingnonanoic acid 0.1% and TFMPVA 0.1%, and shaken-cultured at 30° C. at 125strokes/min. After 24 hours, the cells were collected by centrifugation,resuspended in 200 mL of M9 medium containing TFMPVA 0.2% but not anitrogen source (NH₄Cl), and further shaken-cultured at 30° C. at 125strokes/min. After 24 hours, the cells were collected by centrifugation,washed once with cold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in 100 mL of chloroform and stirredat 60° C. for 20 hours to extract PHA. After the extract was filteredwith a membrane filter of 0.45 μm in pore size, the filtrate wasconcentrated with a rotary evaporator and the concentrate wasreprecipitated in cold methanol. Only the precipitate was then.recovered and vacuum-dried to obtain and weigh PHA. The yields are shownin Table 14.

Evaluation of the molecular weight of the PHA by gel permeationchromatography (GPC: Toso HLC-8020, column: Polymer Laboratory PLgelMIXED-C (5 μm), solvent: chloroform, converted on a polystyrene basis)revealed Mn=64,000 and Mw=110,000.

After the PHA obtained was subjected to methanolysis according to theconventional method, it was analyzed by gas chromatography-spectrometry(GC-MS, Shimadzu QP-5050, EI method, column: DB-WAXETR (30 m×0.32 mm×0.5μm)) and the methyl esters of the PHA monomer unit were identified. TheTIC (total ion chromatogram) and the mass spectrum of a peak (close to36.5′) representing the target unit3-hydroxy-5-(4-trifluoromethylphenyl)valeric acid are shown in FIGS. 12Aand 12B, respectively. The TIC area ratio of each unit of the PHA isshown in Table 15.

The above results indicated that one method of the present inventionproduced PHA containing 3-hydroxy-5-(4-trifluoromethylphenyl)valericacid as a monomer unit.

Example 15

(Production of polymer by strain P91)

Cells of strain P91 were inoculated in 200 mL of M9 medium containingnonanoic acid 0.1% and TFMPVA 0.1%, and shaken-cultured at 30° C. at 125strokes/min. After 30 hours, the cells were collected by centrifugation,resuspended in 200 mL of M9 medium containing TFMPVA 0.1% and nonanoicacid 0.05% but not a nitrogen source (NH₄Cl), and furthershaken-cultured at 30° C. at 125 strokes/min. After 30 hours, the cellswere collected by centrifugation, washed once with cold methanol,lyophilized and weighed.

The lyophilized pellet was suspended in 100 mL of chloroform and stirredat 60° C. for 20 hours to extract PHA. After the extract was filteredwith a membrane filter of 0.45 μm in pore size, the filtrate wasconcentrated with a rotary evaporator and the concentrate reprecipitatedin cold methanol. Only the precipitate was then recovered andvacuum-dried to obtain and weigh PHA. The yield is shown in Table 16.

Evaluation of the molecular weight of the PHA by gel permeationchromatography (GPC; Toso HLC-8020, column: Polymer Laboratory PLgelMIXED-C (5 μm), solvent: chloroform, converted on a polystyrene basis)revealed Mn=69,000 and Mw=120,000.

After the PHA obtained was subjected to methanolysis according to theconventional method, it was analyzed with a gas chromatograph-massspectrometer (GC-MS, Shimadzu QP-5050, EI method, column: DB-WAXETR (30m×0.32 mm×0.5 μm)) and the methyl esters of the PHA monomer unit wereidentified. The TIC (total ion chromatogram) and the mass spectrum of apeak (close to 36.5′) representing the target unit3-hydroxy-5-(4-trifluoromethylphenyl)valeric acid are shown in FIGS. 13Aand 13B, respectively. The TIC area ratio of each unit of the PHA isshown in Table 17.

The above results indicated that one method of the present inventionproduced PHA containing 3-hydroxy-5-(4-trifluoromethylphenyl)valericacid as a monomer unit.

Example 16

Cells of strain YN2 were inoculated in 200 mL of M9 medium containingD-glucose 0.5% or n-nonanoic acid 0.1% and PVA 0.1%, and shaken-culturedat 30° C. at 125 strokes/min. After 40 hours, the cells were collectedby centrifugation, washed once with cold methanol and lyophilized toobtain a lyophilized pellet.

The lyophilized pellet was suspended in 20 mL of chloroform and stirredat 60° C. for 28 hours to extract. PHA. After the extract was filteredwith a membrane filter of 0.45 μm in pore size, the filtrate wasconcentrated with a rotary evaporator and the concentrate wasresuspended in cold methanol. Only the precipitate was then recoveredand vacuum-dried to obtain PHA. After the PHA obtained was subjected tomethanolysis according to the conventional method, it was analyzed witha gas chromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EImethod) and the methyl esters of the PHA monomer unit were identified.As a result, PHA containing 3HPV being the desired PVA-derived monomerunit at a higher ratio was obtained in high yield by using D-glucose asa carbon source for growth as shown in Table 18.

Example 17

Cells of strain YN2 were inoculated in 200 mL of M9 medium containingD-glucose 0.5% or n-nonanoic acid 0.1% and PVA 0.1%, and shaken-culturedat 30° C. at 125 strokes/min. After 48 hours, the cells were collectedby centrifugation, resuspended in 200 mL of M9 medium containingD-glucose 0.5% or n-nonanoic acid 0.1% and PVA 0.1% but not a nitrogensource (NH₄Cl), and further shaken-cultured at 30° C. at 125strokes/min. After 40 hours, the cells were collected by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform, stirred at60° C. for 24 hours to extract PHA. After the extract was filtered witha membrane filter of 0.45 μm in pore size, the filtrate was concentratedwith a rotary evaporator and the concentrate was reprecipitated in coldmethanol. Only the precipitate was then recovered and vacuum-dried toobtain PHA. After the PHA obtained was subjected to methanolysisaccording to the conventional method, it was analyzed with a gaschromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EI method) andthe methyl esters of the PHA monomer unit were identified. As a result,PHA containing 3HPV being the desired PVA-derived monomer unit at ahigher ratio was obtained in a high yield by using D-glucose as a carbonsource for growth, as shown in Table 19.

Example 18

Cells of strain YN2 were inoculated in 200 mL of M9 medium containingD-mannose 0.5% or D-fructose 0.5% and PVA 0.1%, and cultured withshaking at 30° C. at 125 strokes/min. After 100 hours in the D-mannosesystem and 40 hours in the D-fructose system, the cells were collectedby centrifugation, resuspended in 200 mL of M9 medium containingD-mannose 0.5% or D-fructose 0.5% and PVA 0.1% but not a nitrogen source(NH₄Cl), and further shaken-cultured at 30° C. at 125 strokes/min. After48 hours, the cells were collected by centrifugation, washed once withcold methanol and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform and stirredat 60° C. for 24 hours to extract PHA. After the extract was filteredwith a membrane filter of 0.45 μm in pore size, the filtrate wasconcentrated with a rotary evaporator and the concentrate wasreprecipitated in cold methanol. The precipitate was then recovered andvacuum-dried to obtain PHA. After the PHA obtained was subjected tomethanolysis according to the conventional method, it was analyzed witha gas chromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EImethod) and the methyl esters of the PHA monomer unit were identified.As a result, as shown in Table 20, D-mannose and D-fructose were also aseffective as D-glucose as the carbon source to obtain PHA having a highproportion of 3HPV being the desired PVA-derived monomer unit at a highPHA yield.

Example 19

Cells of strain P161 were inoculated in 200 mL of M9 medium containingD-glucose 0.5% or n-nonanoic acid 0.1% and PVA 0.1%, and cultured withshaking at 30° C. at 125 strokes/min. After 48 hours, the cells werecollected by centrifugation, resuspended in 200 mL of M9 mediumcontaining D-glucose 0.5% or n-nonanoic acid 0.1% and PVA 0.1% but not anitrogen source (NH₄Cl), and further cultured at 30° C. at 125strokes/min. After 40 hours, the cells were collected by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform and stirredat 60° C. for 24 hours to extract PHA. After the extract was filteredwith a membrane filter of 0.45 μm in pore size, the filtrate wasconcentrated with a rotary evaporator and the concentrate wasreprecipitated in cold methanol. Only the precipitate was then recoveredand vacuum-dried to obtain PHA. After the PHA obtained was subjected tomethanolysis according to the conventional method, it was analyzed witha gas chromatograph-mass spectrometer (GC-MS, Shimadzu QP-5050, EImethod) and the methyl esters of the PHA monomer unit were identified.As a result, PHA having a higher proportion of 3HPV as the desiredPVA-derived monomer unit was obtained in high yield by using D-glucoseas a carbon source, as shown in Table 21.

Example 20

The cells of strain YN2 were shake-cultured in 200 mL of M9 medium,containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1% of FPVAunder the conditions of 30° C. and 125 strokes/min for 48 hours. Thecells were recovered by centrifugal separation, and resuspended in 200mL of M9 medium, containing 0.5% of D-glucose or 0.1% of n-nonanoic acidand 0.1% of FPVA but no nitrogen source (NH₄Cl), where they were furthershake-cultured under the conditions of 30° C. and 125 strokes/min for 40hours. The cells were recovered by centrifugal separation, washed oncewith cold methanol, and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform, and stirredat 60° C. for 24 hours, to extract the PHA. The extract solution wasfiltered through a membrane having a pore size of 0.45 μm andconcentrated by a rotary evaporator. The concentrated solution wasreprecipitated in cold methanol, and the precipitate only was recoveredand dried under a vacuum, to obtain the PHA. The PHA thus prepared wassubjected to methanolysis by the normal procedure, and analyzed by a gaschromatograph/mass spectrometer (GC-MS, Shimadzu QP-5050, based on theEI method), to identify the methyl-esterified product of the PHA monomerunits. The results are given in Table 22. As shown, culturing withD-glucose as the carbon source for growth gives the PHA having a higherproportion of 3-hydroxy-5-(4-fluorophenyl)valeric acid as the desiredFPVA-derived monomer unit in higher yield.

Example 21

Cells of strain P161 was shake-cultured in 200 mL of M9 medium,containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1% of PHxAunder the conditions of 30° C. and 125 strokes/min for 48 hours. Thecells were recovered by centrifugation, and resuspended in 200 mL of M9medium, containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1%of PHXA but no nitrogen source (NH₄Cl), where they were furthershake-cultured under the conditions of 30° C. and 125 strokes/min for 40hours. The cells were recovered by centrifugation, washed once with coldmethanol, and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform, and stirredat 60° C. for 24 hours, to extract the PHA. The extract solution wasfiltered through a membrane having a pore size of 0.45 μm andconcentrated by a rotary evaporator. The concentrated solution wasreprecipitated in cold methanol, and the precipitate only was recoveredand dried under a vacuum, to obtain the PHA. The PHA thus prepared wassubjected to methanolysis by the normal procedure, and analyzed by a gaschromatograph/mass spectrometer (GC-MS, Shimadzu QP-5050, based on theEI method), to identify the methyl-esterified product of the PHA monomerunits. The results are given in Table 23. As shown, culturing withD-glucose as the carbon source for growth gives the PHA having a higherproportion of 3-hydroxy-6-phenylhexanoic acid as the desiredPHxA-derived monomer unit in higher yield.

Example 22

Cells of strain YN2 were shake-cultured in 200 mL of M9 medium,containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1% of PxBAunder the conditions of 30° C. and 125 strokes/min for 48 hours. Thecells were recovered by centrifugation, and resuspended in 200 mL of M9medium, containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1%of PxBA but no nitrogen source (NH₄Cl), where they were furthershake-cultured under the conditions of 30° C. and 125 strokes/min for 40hours. The cells were recovered by centrifugation, washed once with coldmethanol, and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform, and stirredat 60° C. for 24 hours, to extract the PHA. The extract solution wasfiltered through a membrane having a pore size of 0.45μm andconcentrated by a rotary evaporator. The concentrated solution wasreprecipitated in cold methanol, and the precipitate only was recoveredand dried under a vacuum, to obtain the PHA. The PHA thus prepared wassubjected to methanolysis by the normal procedure, and. analyzed by agas chromatograph/mass spectrometer (GC-MS, Shimadzu QP-5050, based onthe EI method), to identify the methyl-esterified product of the PHAmonomer units. The results are given in Table 24. As shown, culturingwith D-glucose as the carbon source for growth gives the PHA having ahigher proportion of 3-hydroxy-4-phenoxybutyric acid as the desiredPxBA-derived monomer unit in higher yield.

Example 23

Cells of strain H45 was shake-cultured in 200 mL of M9 medium,containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1% of PxBAunder the conditions of 30° C. and 125 strokes/min for 48 hours. Thecells were recovered by centrifugation, and resuspended in 200 mL of M9medium, containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1%of PxBA but no nitrogen source (NH₄Cl), where they were furthershake-cultured under the conditions of 30° C. and 125 strokes/min for 40hours. The cells were recovered by centrifugation, washed once with coldmethanol, and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform, and stirredat 60° C. for 24 hours, to extract the PHA. The extract solution wasfiltered through a membrane having a pore size of 0.45 μm andconcentrated by a rotary evaporator. The concentrated solution wasreprecipitated in Fold methanol, and the precipitate only was recoveredand dried under a vacuum, to obtain the PHA. The PHA thus prepared wassubjected to methanolysis by the normal procedure, and analyzed by a gaschromatograph/mass spectrometer (GC-MS, Shimadzu QP-5050, based on theEI method), to identify the methyl-esterified product of the PHA monomerunits. The results are given in Table 25. As shown, culturing withD-glucose as the carbon source for growth gives the PHA having a higherproportion of 3-hydroxy-4-phenoxybutyric acid as the desiredPxBA-derived monomer unit in higher yield.

Example 24

Cells of strain P161 were shake-cultured in 200 mL of M9 medium,containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1% of PxBAunder the conditions of 30° C. and 125 strokes/min for 48 hours. Thecells were recovered by centrifugation, and resuspended in 200 mL of M9medium, containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1%of PxBA but no nitrogen source (NH₄Cl), where they were furthershake-cultured under the conditions of 30° C. and 125 strokes/min for 40hours. The cells were recovered by centrifugation, washed once with coldmethanol, and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform, and stirredat 60° C. for 24 hours, to extract the PHA. The extract solution wasfiltered through a membrane having a pore size of 0.45 μm andconcentrated by a rotary evaporator. The concentrated solution wasreprecipitated in cold methanol, and the precipitate only was recoveredand dried under a vacuum, to obtain the PHA. The PHA thus prepared wassubjected to methanolysis by the normal procedure, and analyzed by a gaschromatograph/mass spectrometer (GC-MS, Shimadzu QP-5050, based on theEI method), to identify the methyl-esterified product of the PHA monomerunits. The results are given in Table 26. As shown, culturing withD-glucose as the carbon source for growth gives the PHA having a higherproportion of 3-hydroxy-4-phenoxybutyric acid as the desiredPxBA-derived monomer unit in higher yield.

Example 25

Cells of strain YN2 were shake-cultured in 200 mL of M9 medium,containing 0.5% of D-glucose or 0.1% of n-nonanoic acid and 0.1% of CHBAunder the conditions of 30° C. and 125 strokes/min for 40 hours. Thecells were recovered by centrifugation, washed once with cold methanol,and lyophilized.

The lyophilized pellet was suspended in 20 mL of chloroform, and stirredat 60° C. for 28 hours, to extract the PHA. The extract solution wasfiltered through a membrane having a pore size of 0.45 μm andconcentrated by a rotary evaporator. The concentrated solution wasreprecipitated in cold methanol, and the precipitate only was recoveredand dried under a vacuum, to obtain the PHA. The PHA thus prepared wassubjected to methanolysis by the normal procedure, and analyzed by a gaschromatograph/mass spectrometer (GC-MS, Shimadzu QP-5050, based on theEI method), to identify the methyl-esterified product of the PHA monomerunits. The results are given in Table 27. As shown, culturing withD-glucose as the carbon source for growth gives the PHA having a higherproportion of 3HCHB as the desired CHBA-derived monomer unit in higheryield.

Example 26

(Production of Poly-3-hydroxy-5-phenylvaleric Acid by Strain YN2)

A colony of strain YN2 on M9 agar medium containing 0.1% of nonanoicacid (hereinafter referred as to NA) was inoculated in a total of 4types of media (each 200 mL), (1) M9 liquid medium containing 0.5% of ayeast extract (DIFCO, hereinafter referred to as YE) and 0.1% of5-phenylvaleric acid, (2) M9 liquid medium containing 0.5% of a beefextract (DIFCO, hereinafter referred to as BE) and 0.1% of5-phenylvaleric acid, (3) M9 liquid medium containing 0.5% of Casaminoacid (DIFCO, hereinafter referred to as CA) and 0.1% of 5-phenylvalericacid, and (4) M9 liquid medium containing 0.5% of polypeptone (WakoJun-yaku, hereinafter referred to as PP) and 0.1% of 5-phenylvalericacid, and cultured at 30° C. for 24 hours. The cells were recovered fromeach medium by centrifugation, washed once with cold methanol, andlyophilized.

The lyophilized pellets from each medium were weighed and suspended in100 mL of chloroform, and stirred at 55° C. for 20 hours, to extract thePHA. Each extract solution was filtered through a membrane having a poresize of 0.45 μm and concentrated by a evaporator. The concentratedsolution was reprecipitated in cold methanol, to obtain the polymer,which was dried under a vacuum at room temperature and weighed. Theyield results are given in Table 28.

The PHA composition thus prepared was analyzed by the followingprocedure. Approximately 10 mg of the PHA was dissolved in 2 mL ofchloroform in a 25 mL egg-plant type flask, to which 2 mL of a methanolsolution containing 3% sulfuric acid was added. The mixture was heatedat 100° C. with reflux for 3.5 hours for the reactions. On completion ofthe reactions, the effluent was incorporated with 10 mL of deionizedwater and vigorously shaken for 10 min. It was separated into twolayers, and the lower chloroform layer was taken out, dehydrated withmagnesium sulfate, and analyzed by a gas chromatograph/mass spectrometer(GC-MS, Shimadzu QP-5050, based on the EI method, with a 0.32 mm by 30 mcolumn (J&W, DB-WAX)), to identify the methyl-esterified product of thePHA monomer units. The PHA monomer units were found to comprise 96% of3-hydroxy-5-phenylvaleric acid unit and 4% of 3-hydroxybutyric acidunit.

The results indicate that one of the embodiments of the presentinvention gives the PHA containing a very high proportion of the3-hydroxy-5-phenylvaleric acid unit in a high yield.

Example 27

(Production of Poly-3-hydroxy-4-cyclohexylbutyric Acid by Strain YN2)

A colony of strain YN2 grown on M9 agar medium containing 0.1% of NA wasinoculated into 2 types of media (each 200 mL), (1) M9 liquid mediumcontaining 0.5% of YE and 0.1% of 4-cyclohexylbutyric acid, and (2) M9liquid medium containing 0.5% of PP and 0.1% of 4-cyclohexylbutyricacid, and cultured at 30° C. for 24 hours. The cells were recovered fromeach medium by centrifugation, washed once with cold methanol, andlyophilized.

The lyophilized pellets from each medium were weighed and suspended in100 mL of chloroform, and stirred at 55° C. for 20 hours, to extract thePHA. Each extract solution was filtered through a membrane having a poresize of 0.45 μm and concentrated by a evaporator. The concentratedsolution was reprecipitated in cold methanol, to obtain the polymer,which was dried under a vacuum at room temperature and weighed. Theyield results are given in Table 29.

The PHA composition thus prepared was analyzed in a manner similar tothat for EXAMPLE 26. The PHA monomer units were found to comprise 97% of3-hydroxy-4-cyclohexylbutyric acid unit and 3% of 3-hydroxybutyric acidunit.

The results indicate that one of the embodiments of the presentinvention gives the PHA containing a very high proportion of the3-hydroxy-4-cyclohexylbutyric acid unit in a high yield.

Example 28

(Production of Poly-3-hydroxy-5-phenoxyvaleric Acid by Strain YN2)

A colony of strain YN2 grown on M9 agar medium containing 0.1% of NA wasinoculated in 2 types of media (each 200 mL), (1) M9 liquid mediumcontaining 0.5% of YE and 0.1% of 5-phenoxyvaleric acid, and (2) M9liquid medium containing 0.5% of PP and 0.1% of 5-phenoxyvaleric acid,and cultured at 30° C. for 26 hours. The cells were recovered from eachmedium by centrifugation, washed once with cold methanol, andlyophilized.

The lyophilized pellets from each medium were weighed and suspended in100 mL of chloroform, and stirred at 55° C. for 20 hours, to extract thePHA. Each extract solution was filtered through a membrane having a poresize of 0.45 μm and concentrated by a evaporator. The concentratedsolution was reprecipitated in cold methanol, to obtain the polymer,which was dried under a vacuum at room temperature and weighed. Theyield results are given in Table 30.

The PHA composition thus prepared was analyzed in a manner similar tothat in EXAMPLE 26. The PHA monomer units were found to comprise 95% of3-hydroxy-5-phenoxyvaleric acid unit and 5% of 3-hydroxybutyric acidunit.

The results indicate that one of the embodiments of the presentinvention gives the PHA containing a very high proportion of the3-hydroxy-5-phenoxyvaleric acid unit in a high yield.

Example 29

(Production of Poly-3-hydroxy-5-phenylvaleric Acid by Strain H45)

A colony of strain H45 grown on M9 agar medium containing 0.1% of NA wasinoculated in 4 types of media (each 200 mL), (1) M9 liquid mediumcontaining 0.5% of YE and 0.1% of 5-phenylvaleric acid, (2) M9 liquidmedium containing 0.5% of sodium glutamate (Kishida Kagaku, hereinafterreferred to as SG) and 0.1% of 5-phenylvaleric acid, (3) M9 liquidmedium containing 0.5% of CA and 0.1% of 5-phenylvaleric acid, and (4)M9 liquid medium containing 0.5% of PP and 0.1% of 5-phenylvaleric acid,and cultured at.30° C. for 28 hours. The cells were recovered from eachmedium by centrifugation, washed once with cold methanol, andlyophilized.

The lyophilized pellets from each medium were weighed and suspended in100 mL of chloroform, and stirred at 55° C. for 20 hours, to extract thePHA. Each extract solution was filtered through a membrane having a poresize of 0.45 μm and concentrated by a evaporator. The concentratedsolution was reprecipitated in cold methanol, to obtain the polymer,which was dried under a vacuum at room temperature and weighed. Theyield results are given in Table 31.

The PHA composition thus prepared was analyzed in a manner similar tothat in EXAMPLE 26. The PHA was found to be essentially the homopolymerof 3-hydroxy-5-phenylvaleric acid.

The results indicate that one of the embodiments of the presentinvention gives the PHA containing a very high proportion of the3-hydroxy-5-phenylvaleric acid unit in a high yield.

Example 30

(Production of Poly-3-hydroxy-5-phenylvaleric Acid by Strain P161)

A colony of strain P161 grown on M9 agar medium containing 0.1% of NAwas inoculated in 4 types of media (each 200 mL), (1) M9 liquid mediumcontaining 0.5% of YE and 0.1% of 5-phenylvaleric acid, (2) M9 liquidmedium containing 0.5% of SG and 0.1% of 5-phenylvaleric acid, (3) M9liquid medium containing 0.5% of BE and 0.1% of 5-phenylvaleric acid,and (4) M9 liquid medium containing 0.5% of PP and 0.1% of5-phenylvaleric acid, and cultured at 30° C. for 24 hours. The cellswere recovered from each medium by centrifugation, washed once with coldmethanol, and lyophilized.

The lyophilized pellets from each medium were weighed and suspended in100 mL of chloroform, and stirred at 55° C. for 20 hours, to extract thePHA. Each extract solution was filtered through a membrane having a poresize of 0.45 μm and concentrated by a evaporator. The concentratedsolution was reprecipitated in cold methanol, to obtain the polymer,which was dried under a vacuum at room temperature and weighed. Theyield results are given in Table 32.

The PHA composition thus prepared was analyzed in a manner similar tothat in EXAMPLE 26. The PHA monomer units were found to comprise 97% of3-hydroxy-5-phenylvaleric acid unit and 3% of 3-hydroxybutyric acidunit.

The results indicate that one of the embodiments of the presentinvention gives the PHA containing a very high proportion of the3-hydroxy-5-phenylvaleric acid unit in a high yield.

Example 31

Pseudomonas cichorii strain YN2 was shake-cultured in 10 mL of M9 mediumcontaining 0.5% of D-glucose under the conditions of 30C and 125strokes/min for 72 hours, and 2 mL of the culture was transferred forfurther shake-culture in 200 mL of M9 medium containing 0.5% ofD-glucose and 0.1% of N0₂PxBA under the conditions of 30° C. and 125strokes/min for another 72 hours. The cells were recovered bycentrifugation, and resuspended in 200 mL of M9 medium, containing 0.5%of D-glucose and 0.1% of NO₂PxBA but no nitrogen source (NH₄Cl), wherethey were further shake-cultured under the conditions of 30° C. and 125strokes/min for 48 hours. The cells were recovered by centrifugation,washed once with cold methanol, and lyophilized.

The lyophilized pellets were suspended in 20 mL of chloroform, andstirred at 60° C. for 20 hours, to extract the PHA. The extract solutionwas filtered through a membrane having a pore size of 0.45 μm andconcentrated by a rotary evaporator. The concentrated solution wasreprecipitated in cold methanol, and the precipitate only was recoveredand dried under a vacuum, to obtain the PHA. The PHA thus prepared wasanalyzed by NMR under the following conditions:

<Analyzer>

FT-NMR: Bruker DPX400

Resonance frequency: ¹H=400 MHz

<Analysis conditions>

Nuclide to be analyzed: ¹H

Solvent: CDCl₃

Reference: TMS/CDCl₃ sealed in a capillary

Temperature: room temperature

FIG. 14 shows the ¹H-NMR spectral patterns, Table 33 the results ofidentification of the patterns, and Table 34 the composition of themonomer units of 3-hydroxy-4-(4-nitrophenoxy)butyric acid. As shown inTable 33, the PHA is the one represented by the chemical formula (45),containing 3-hydroxy-4-(4-nitrophenoxy)butyric acid as the monomer unit.

The PHA thus prepared was subjected to methanolysis by the normalprocedure, and analyzed by a gas chromatograph/mass spectrometer (GC-MS,Shimadzu QP-5050, based on the EI method), to identify themethyl-esterified product of the PHA monomer units, other than3-hydroxy-4-(4-nitrophenoxy)butyric acid. The results are given in Table34.

The PHA had a number-average molecular weight (Mn) of 81,900 andweight-average molecular weight (Mw) of 226,200, as determined by a gelpermeation chromatograph (GPC; Toso HLC-8020, column: PolymerLaboratory's PLgel MIXED-C(5 μm), solvent: chloroform, as polystyrene).

Example 32

Cells of Pseudomonas cichorii strain YN2 were inoculated into 200 mL ofM9 culture medium containing 0.5% D-glucose and 0.1% NO₂PxBA, and shakecultured at 30° C. at 125 stroke/min. After 45 hours, the cells wererecovered by centrifugation and re-suspended into 200 mL of M9 culturemedium containing 0.5% D-glucose, 0.1% NO₂PxBA and nitrogen(NH₄Cl)-free, and further shake cultured at 30° C. at 125 stroke/min.After 48 hours, the cells were recovered by centrifugation, washed oncewith cold methanol and lyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by a rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis under the conditionshown in the Example 31. As a result, it was confirmed that the PHA wasthe PHA containing 3-hydroxy-4-(4-nitorophenoxy)butyric acid as amonomer unit, as shown in Table 35.

Further, the resulting PHA was done methanolysis according toconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(4-nitrophenoxy)butyric acid. The result wasshown in the Table 35.

Example 33

Cells of Pseudomonas cichorii strain YN2 were inoculated into 200 mL ofM9 culture medium containing 0.5% Polypeptone and 0.1% NO₂PxBA, andshake cultured at 30° C. at 125 stroke/min. After 21 hours, the cellswere recovered by centrifugation and re-suspended into 200 mL of M9culture medium containing 0.5% sodium pyruvate, 0.1% NO₂PxBA andnitrogen (NH₄Cl)-free, and further shake cultured at 30° C. at 125stroke/min. After 24 hours, the cells were recovered by centrifugation,washed once with cold methanol and lyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis under the conditionshown in the Example 31. As a result, it was confirmed that the PHA wasthe PHA containing 3-hydroxy-4-(4-nitorophenoxy)butyric acid as amonomer unit, as shown. in Table 36.

Further, the resulting PHA was done methanolysis according to theconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(4-nitrophenoxy)butyric acid. The result wasshown in the Table 36.

Example 34

First, cells of Pseudomonas cichorii strain YN2 were inoculated into 10mL of M9 culture medium containing 0.5% D-glucose and shake-cultured at30° C. at 125 stroke/min, and then 2 mL of the culture was added into200 mL of M9 culture medium containing 0.5% D-glucose and 0.1% CNPxBA,and shake cultured at 30° C. at 125 stroke/min. 48 hours later, thecells were recovered by centrifugation and re-suspended into 200 mL ofM9 culture medium containing 0.5% D-glucose, 0.1% CNPxBA and nitrogen(NH₄Cl)-free, and further shake cultured at 30° C. at 125 stroke/min. 47hours later, the cells were recovered by centrifugation, washed oncewith cold methanol and lyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis on the followingconditions.

<Measuring apparatus>

FT-NMR: Bruker DPX 400

Resonance frequency: ¹H=400 MHz

<Measuring condition>

Measuring nuclide: ¹H

Measuring solvent: CDCl₃

Reference: capillary inclusion TMS/CDCl₃

Measuring temperature: room temperature

FIG. 15 illustrates ¹H-NMR spectra, Table 37 shows their correspondingresults, and Table 38 shows the ratio of3-hydroxy-4-(4-cyanophenoxy)butyric acid monomer unit contained in PHA.As shown in the Table 37, it was confirmed that the PHA was the PHAexpressed by the chemical formula (46) containing3-hydroxy-4-(4-cyanophenoxy)butyric acid as a monomer unit.

Further, the resulting PHA was done methanolysis according to theconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(⁴-cyanophenoxy)butyric acid. The result wasshown in the Table 38.

Moreover, the molecular weight of the PHA was evaluated by gelpermeation chromatography (GPC; Toso HLC-8020, column; PolymerLaboratory PL gel MIXED-C (5 μm), solvent; (chloroform, polystyrenereduced) to obtain Mn=58200 and Mw=108100.

Example 35

First, cells of Pseudomonas cichorii strain H45 were inoculated into 10mL of M9 culture medium containing 0.5% D-glucose and shake-cultured at30° C. at 125 stroke/min, and then 2 mL of the culture were added into200 mL of M9 culture medium containing 0.5% D-glucose and 0.1% CNPxBA,and shake cultured at 30° C. at 125 stroke/min. 48 hours later, thecells were recovered by centrifugation and re-suspended into 200 mL ofM9 culture medium containing 0.5% D-glucose, 0.1% CNPxBA and nitrogen(NH₄Cl)-free, and further shake cultured at 30° C. at 125 stroke/min. 47hours later, the cells were recovered by centrifugation, washed oncewith cold methanol and lyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis under the conditionshown in the Example 34. As a result, it was confirmed that the PHA wasthe PHA containing 3-hydroxy-4-(4-cyanophenoxy)butyric acid as a monomerunit, as shown in Table 39.

Further, the resulting PHA was done methanolysis according to theconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(4-cyanophenoxy)butyric acid. The result wasshown in the Table 39.

Example 36

Cells of Pseudomonas cichorii strain YN2 were inoculated into 200 mL ofM9 culture medium containing 0.5% D-glucose and 0.1% CNPxBA, and shakecultured at 30° C. at 125 stroke/min. After 48 hours, the cells wererecovered by centrifugation and re-suspended into 200 mL of M9 culturemedium containing 0.5% D-glucose, 0.1% CNPxBA and nitrogen (NH₄Cl)-free,and further shake cultured at 30° C. at 125 stroke/min. After 48 hours,the cells were recovered by centrifugation, washed once with coldmethanol and lyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis under the conditionshown in the Example 34. As a result, it was confirmed that the PHA wasthe PHA containing 3-hydroxy-4-(4-cyanophenoxy)butyric acid as a monomerunit, as shown in Table 40.

Further, the resulting PHA was done methanolysis according to theconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(4-cyanophenoxy)butyric acid. The result wasshown in the Table 40.

Example 37

Cells of Pseudomonas cichorii strain H45 were inoculated into 200 mL ofM9 culture medium containing 0.5% D-glucose and 0.1% CNPxBA, and shakecultured at 30° C. at 125 stroke/min. After 48 hours, the cells wererecovered by centrifugation and re-suspended into 200 mL of M9 culturemedium containing 0.5% D-glucose, 0.1% CNPxBA and nitrogen (NH₄Cl)-free,and further shake cultured at 30° C. at 125 stroke/min. After 48 hours,the cells were recovered by centrifugation, washed once with coldmethanol and lyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis under the conditionshown in the Example 34. As a result, it was confirmed that the PHA wasthe PHA containing 3-hydroxy-4-(4-cyanophenoxy)butyric acid as a monomerunit, as shown in Table 41.

Further, the resulting PHA was done methanolysis according to theconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(4-cyanophenoxy)butyric acid. The result wasshown in the Table 41.

Example 38

Cells of Pseudomonas Cichorii strain YN2 were inoculated into 200 mL ofM9 culture medium containing 0.5% polypeptone and 0.1% CNPxBA andcultured at 30° C. with shaking at 125 stroke/min. After 23 hours, thecells were recovered by centrifugation and re-suspended into 200 mL ofM9 culture medium containing 0.5% sodium pyruvate, 0.1% CNPxBA but nonitrogen source (NH₄Cl), and further cultured at 30° C. at 125stroke/min. After 24 hours, the cells were recovered by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis under the conditionshown in the Example 34. As a result, it was confirmed that the PHA wasthe PHA containing 3-hydroxy-4-(4-cyanophenoxy)butyric acid as a monomerunit, as shown in Table 42.

Further, the resulting PHA was done methanolysis according to theconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(4-cyanophenoxy)butyric acid. The result wasshown in the Table 42.

Example 39

Cells of Pseudomonas cichorii strain H45 were inoculated into 200 mL ofM9 culture medium containing 0.5% polypeptone and 0.1% CNPxBA, andcultured at 30° C. with shaking at 125 stroke/min. After 23 hours, thecells were recovered by centrifugation and re-suspended into 200 mL ofM9 culture medium containing 0.5% Sodium pyruvate, 0.1% CNPxBA but nonitrogen source (NH₄Cl), and further shake cultured at 30° C. at 125stroke/min. After 24 hours, the cells were recovered by centrifugation,washed once with cold methanol and lyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was determined by NMR analysis under the conditionshown in the Example 34. As a result, it was confirmed that the PHA wasthe PHA containing 3-hydroxy-4-(4-cyanophenoxy)butyric acid as a monomerunit, as shown in Table 43.

Further, the resulting PHA was done methanolysis according to theconventional method, and then analyzed by gas chromatography-massspectrometer (GC-MS, Shimadzu QP-5050, EI method) to identifymethylesterified materials of PHA monomer unit and identify monomer unitother than 3-hydroxy-4-(4-cyanophenoxy)butyric acid. The result wasshown in the Table 43.

Example 40

(Synthesis of 4-(4-Fluorophenoxy)butyric Acid)

A new compound, 4-(4-fluorophenoxy)butyric acid was prepared by thesynthetic method mentioned below.

Into a round-bottom flask with four opening was put 240 mL of dehydratedacetone, added 15.2 g (0.11 mol) of potassium carbonate, and stirredunder nitrogen atmosphere. Into this solution were added 9.0 g (0.06mol) of sodium iodide and 7.9 g (0.07 mol) of 4-fluorophenol, andthoroughly stirred at room temperature under nitrogen atmosphere. 11.7 g(0.06 mol) of 4-bromoethyl butyrate was then added, and heat refluxed at65° C. for 24 hours.

After aforesaid reaction was completed, the solvent acetone was removedby rotating evaporator, and its residue was re-dissolved in chloroform.Water was added for phase separation, and the organic layer wascollected. After the organic layer was dehydrated with anhydrousmagnesium sulfate, chloroform was removed by rotating evaporator. Then,it was dried by a vacuum pump to provide 14.0 g of crude4-(4-fluorophenoxy)ethyl butyrate (gas chromatograph-mass spectrometer:GC-MS Shimadzu QP-5050, GC-MS peak ratio purity: 65.2% with EI method).Without purifying the resulting crude 4-(4-fluorophenoxy)ethyl butyrate,it was used in the following ester hydrolytic reaction.

The resulting crude 4-(4-fluorophenoxy)ethyl butyrate was dissolved in300 mL of ethanol-water (1:9 (V/V)) mixed solution, and approximatelyten-fold molar equivalent weight of potassium hydroxide was added toreact for four hours under ice cooling (at 0 to 4° C.). This cocktailwas poured into 3 L of 0.1 mol/L hydrochloric acid to precipitate.Precipitates were filtrated, separated, and dried by vacuum pump toprovide crude 4-(4-fluorophenoxy)butyric acid.

The resulting crude 4-(4-fluorophenoxy)butyric acid (precipitate) wasdissolved in a small amount of hot methanol, and gradually cooled torecrystallize. Filtrated recrystallization materials were dried byvacuum pump to provide objective compound, 4-(4-fluorophenoxy)butyricacid. In this series of processes, the resulting4-(4-fluorophenoxy)butyric acid was 52.7% in yield based on the rawmaterial 4-bromoethylbutyrate.

In order to verify that the resulting compound was the objective4-(4-fluorophenoxy)butyric acid, NMR analysis was carried out by thefollowing measuring apparatus under measuring condition to identify thestructure.

<Measuring apparatus>

FT-NMR: Bruker DPX 400

<Measuring condition>

Resonance frequency: ¹H 400 MHz

¹³C 100 MHz

Measuring nuclide: ¹H, ¹³C

Used solvent: CDCl₃

Reference: capillary inclusion TMS/CDCl₃

Measuring temperature: room temperature

Determined ¹H-NMR spectra chart and ¹³C-NMR spectra chart wereillustrated in FIGS. 16 and 17, respectively. Table 44 and 45 showanalytical (corresponding) result of each signal for NMR spectraillustrated in the FIGS. 16 and 17. This analytical (corresponding)result confirmed that the resulting compound was the objective4-(4-fluorophenoxy)butyric acid.

Example 41

First, cells of Pseudomonas cichorii strain YN2 (FERM BP-7375) wereinoculated into 10 mL of M9 culture medium containing 0.5% D-glucose,and shake cultured at 30° C. at 125 stroke/min for 72 hours. Then 2 mLof the culture was added into 200 mL of M9 culture medium (no inorganicnitrogen source, NH₄Cl) containing 0.5% D-glucose and 0.1% pFPxBA, andcontinuously shake cultured at 125 stroke/min. After 45 hours, the cellswere recovered by centrifugation and re-suspended into 200 mL of M9culture medium containing 0.5% D-glucose and 0.1% pFPxBA, and furthershake cultured at 30° C. at 125 stroke/min. After 46 hours, the cellswere recovered by centrifugation, washed once with cold methanol andlyophilized.

This lyophilized pellet was suspended into 20 mL of chloroform, andstirred at 60° C. for 20 hours to extract PHA. After the extracts werefiltrated through a membrane filter with a pore size of 0.45 μm, theywere concentrated by rotating evaporator, and the concentrates werere-precipitated with cold methanol, and further the precipitates alonewere recovered and vacuum dried to provide PHA.

The resulting PHA was done methanolysis according to the conventionalmethod, and then analyzed by gas chromatography-mass spectrometer(GC-MS, Shimadzu QP-5050, EI method) to identify methylesterifiedmaterials of PHA monomer unit. FIG. 18 illustrates measured GC-MSspectra data, and the upper chart shows GC spectrum, and the lower chartshows an MS spectrum corresponding to the main peak on the GC spectrum.This result shows that the resulting PHA contains3-hydroxy-4-(4-fluorophenoxy)butyric acid (3HpFPxB) as a main componentof monomer unit, in addition it also contains a small amount of sixkinds of monomer units, and can be represented by the following chemicalformula (47).

Furthermore, the molecular weight of the PHA was determined by gelpermeation chromatography (GPC; Toso.HLC-8020, column; Polymerlaboratory.PL gel.MIX ED-C-5 μm, solvent; chloroform, polystyrenereduced molecular weight).

Identification result, average molecular weight, as well as yield oflyophilized pellet and recovered polymer are shown in Table 46. It showsthat the resulting PHA is the PHA containing3-hydroxy-4-(4-fluorophenoxy)butyric acid (3HpFPxB) as a monomer unit.Further, it is likely that the extracted PHA includes 3HpFPxB unit as amajor component, however, it is the mixture containing more than onekind of component as a monomer unit selected from the group consistingof 3-hydroxybutyric acid, 3-hydorxyhexanoic acid, 3-hydroxyoctanoicacid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid,3-hydroxydodecenoic acid. Estimated average molecular weight wasMn=42400 for number-average molecular weight, on the other hand,Mw=90600 for weight-average molecular weight.

This PHA was also determined by NMR analysis with the same measuringapparatus and same measuring condition as shown in the Example 40. Themeasured ¹H-NMR spectra chart is shown in FIG. 19. Table 47 shows theanalytical (corresponding) result of each signal for major peak for NMRspectra illustrated in the FIG. 19. This analytical (corresponding)result confirmed that the resulting PHA contained 3HpFPxB unit as amajor component.

Example 42

Cells of Pseudomonas cichorii H45; FERM BP-7374 were inoculated in 10 mLof M9 medium containing D-glucose 0.5%, and was shake-cultured at 30° C.in 125 strokes/min. for 72 hours. Then 2 ml of the culture wastransferred into 200 ml of M9 medium containing 0.5% D-glucose and 0.1%pFPxBA, and further shake cultured at 30° C. in 125 strokes/min. After45 hours, cultured cells were recovered by centrifugation, andre-suspended in M9 medium containing D-glucose 0.5% and pFPxBA 0.1%, butnot containing inorganic nitrogen source (NH₄Cl), and further culturedat 30° C. in 125 strokes/min. After 46 hours, cells were recovered bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated. the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 48, result of identification, and, weight obtained and yield ofthe lyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA with 3HpFPxB as a monomer unit. The extractedPHA mainly consists of 3HpFPxB as a main component, and is considered tobe a mixture containing more than one of compounds selected from thegroup consisting of 3-hydroxybutyric acid, 3-hydroxyhexanoic acid,3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, 3-hydroxydodecanoic acidand 3-hydroxydodecenoic acid, as a monomer unit.

Example 43

Cells of Pseudomonas cichorii YN2 were inoculated in 200 ml M9 mediumcontaining D-glucose 0.5% and pFPxBA 0.1% and was shake cultured at 30°C. in 125 strokes/min. After 96 hours, the cultured cells were recoveredby centrifugation, and re-suspended in 200 ml of M9 medium containingD-glucose 0.5% and pFPxBA 0.1% but not containing an inorganic nitrogensource (NH₄Cl), and further shake cultured at 30° C. in 125 strokes/min.After 64 hours, cells were recovered by centrifugation, washed once withcold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 49, result of identification, and weight obtained and yield of thelyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA with 3HpFPxB as a major monomer unit.

Example 44

Cells of Pseudomonas cichorii H45 were inoculated in M9 medium 200 mLcontaining D-glucose 0.5% and pFPxBA 0.1% and was shake cultured at 30°C. in 125 strokes/min. After 96 hours, cultured cells were recovered bycentrifugation, and re-suspended in M9 medium not containing inorganicnitrogen source NH₄Cl, containing D-glucose 0.5% and pFPxBA 0.1% (200ml), then further shake cultured at 30° C. in 125 strokes/min. After 64hours, cells were recovered by centrifugation, washed once with coldmethanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter of pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 50, result of identification, and, weight obtained and yield ofthe lyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA with 3HpFPxB as a major monomer unit.

Example 45

Cells of Pseudomonas cichorii YN2 were inoculated in 200 ml of M9 mediumcontaining polypeptone 0.5% and pFPxBA 0.1% and was shake cultured at30° C. in 125 strokes/min. After 24 hours, cultured cells were recoveredby centrifugation, and re-suspended in 200 ml of M9 medium containingsodium pyruvate 0.5% and pFPxBA 0.1%, but not containing inorganicnitrogen source NH₄Cl, and further shake cultured at 30° C. in 125strokes/min. After 24 hours, cells were recovered by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 2b hours to extract PHA. The extract was filtered usingmembrane filter (pore size 0.45 μm), concentrated by rotary evaporator,precipitated the concentrate with cold methanol, and recovered theprecipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 51, result of identification, weight obtained and yield of thelyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA with 3HpFPxB as a major monomer unit.

Example 46

Cells of Pseudomonas cichorii H45 were inoculated in 200 ml of M9 mediumcontaining polypeptone 0.5% and pFPxBA 0.1% and was shake cultured at30° C. in 125 strokes/min. After 24 hours, the cells were recovered bycentrifugation, and re-suspended in 200 ml of M9 medium not containinginorganic nitrogen source NH₄Cl, and containing sodium pyruvate 0.5% andpFPxBA 0.1%, then further shake cultured at 30° C. in 125 strokes/min.After 24 hours, the cells were recovered by centrifugation, washed oncewith cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 52, result of identification, and, weight obtained and yield ofthe lyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA with 3HpFPxB as a major monomer unit.

Example 47

(Synthesis of 4-(3-Fluorophenoxy)butyric Acid)

Novel compound 4-(3-fluorophenoxy)butyric acid was prepared by thefollowing synthetic method.

Dehydrated acetone 240 mL was poured in a four-neck round-bottom flask,and potassium carbonate 15.2 g (0.11 mol) was added thereto, thenstirred under nitrogen atmosphere. Sodium iodide 9.0 g (0.06 mol) and3-fluorophenol 7.9 g (0.07 mol) were added to the solution, and themixture was stirred sufficiently at room temperature under nitrogenatmosphere. Then ethyl 4-bromobutyrate 11.7 g (0.06 mol) and refluxed at65° C. for 24 hours.

After completed the above reaction, solvent acetone was distilled offusing rotary evaporator, and the residue was dissolved in chloroform.Water was added therein for separation and the organic layer wascollected. The organic layer was dehydrated by adding anhydrousmagnesium sulfate and the chloroform was distilled off by using rotaryevaporator. Further, the residue was dried in vacuo using vacuum pump toobtain crude ethyl 4-(3-fluorophenoxy) butyrate 14 g (purification87.9%, GC-MS peak ratio, determined by EI method using gaschromatography-mass spectrograph: GC-MS Shimadzu QP-5050). The thusobtained crude ethyl 4-(3-fluorophenoxy) butyrate was used subsequentester hydrolysis without purification.

The thus obtained crude ester 3.0 g was dissolved in a mixture ofethanol-water [1:9 (V/V)] 100 mL, to which about 10-fold excess molarequivalent of potassium hydroxide was added, and the mixture was reactedat room temperature for 4 hours. The reaction mixture was poured intoabout 200 mL of 0.1 mol/l aqueous hydrochloric acid to precipitate. Theprecipitate was filtered, separated and dried in vacuo under the vacuumpump to obtain crude 4-(3-fluorophenoxy) butyric acid.

The thus obtained crude 4-(3-fluorophenoxy) butyric acid (precipitate)was dissolved in a small amount of hot methanol and gradually cooled forrecrystallization. Filtered recrystallized product was dried usingvacuum pump to obtain 4-(3-fluorophenoxy) butyric acid.

The yield of 4-(3-fluorophenoxy) butyric acid obtained from crude ester3.0 g was 2.4 g. Consequently, total yield in the whole process based onthe raw material ethyl 4-bromobutyrate is 93.2%.

For verification of the objective compound 4-(3-fluorophenoxy) butyricacid, structure of the obtained compound was identified by NMR usingfollowing measuring apparatus and conditions.

<Measuring apparatus>

FT-NMR: Bruker DPX400

<Measuring condition>

Resonance frequency: ¹H 400 MHZ

¹³C 100 MHZ

Measuring nuclide: ¹H and ¹³C

Solvent used: CDCl₃

Reference: Capillary sealed TMS/CDCl₃

Measuring temperature: room temperature

¹H-NMR spectrum and ¹³C-NMR spectrum are shown in FIG. 20 and FIG. 21,respectively. In Table 53 and Table 54, results of analyses(assignments) of respective signals of NMR spectra, which are shown inFIG. 20 and FIG. 21, are shown. According to the result of analysis(assignment), the obtained compound is confirmed to be the objectivecompound 4-(3-fluorophenoxy) butyric acid.

Example 48

Cells of Pseudomonas cichorii YN2; FERM BP-7375 were inoculated in 10 mLof M9 medium containing D-glucose 0.5% and shake cultured at 30° C. at125 strokes/min for 72 hours. Then 2 mL of the culture was transferredinto 200 ml of M9 medium containing D-glucose 0.5% and mFPxBA 0.1% andshake cultured at 125 strokes/min. After 45 hours, the cells wererecovered by centrifugation, and re-suspended in 200 ml of M9 mediumcontaining D-glucose 0.5% and mFPxBA 0.1% but not containing inorganicnitrogen source NH₄Cl, and further shake cultured at 30° C. at 125strokes/min. After 47 hours, cells were recovered by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InFIG. 22, measured GC-MS spectrum data is shown. Upper part of FIG. 22indicates GC spectrum, and the lower part indicates MS spectrumcorresponding to main peaks on the above GC spectrum. As a result, thethus obtained PHA is the PHA, which contains3-hydroxy-4-(3-fluorophenoxy) butyric acid (3HmFPxB) as a major monomerunit as well as small amount of 6 types of monomer units, and thecompound can be illustrated as the following chemical structure (48).

Molecular weight of PHA was measured by using gel-permeationchromatography (GPC: Toso HLC-8020; Column: Polymer Laboratory PLgelMIXED-C, 5 μm; Solvent: chloroform: Molecular weight: reduced value forpolystyrene).

In Table 55, result of identification, average molecular weight, andweight obtained and yield of the lyophilized pellet and the recoveredpolymer are shown. The thus obtained PHA is the PHA, which contains3-hydroxy-4-(3-fluorophenoxy) butyric acid (3HmFPxB) as a monomer unit.The extracted PHA mainly consists of 3HmFPxB unit as a main component,and is considered to be a mixture containing more than one of compoundsselected from the group consisting of 3-hydroxybutyric acid,3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid,3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid, as a monomerunit. The evaluated average molecular weight is: number averagemolecular weight Mn=34500 and weight-average molecular weight Mw=75200.

NMR analysis of the PHA was performed by the same procedures using samemeasuring apparatus and measuring conditions as described in example 47.Measured ¹H-NMR spectrum is shown in FIG. 23. In Table 56, result ofanalysis (assignment) of major peak signals of NMR spectrum in FIG. 23is shown. According to the result of analysis (assignment), the obtainedcompound is confirmed to have 3HmFPxB unit as a major component.

Example 49

Cells of Pseudomonas cichorii H45; FERM BP-7374 were inoculated in 10 mLof M9 medium containing D-glucose 0.5% and was cultured at 30° C. withshaking at 125 strokes/min for 72 hours. Then 2 mL of the culture wastransferred into 200 mL of M9 medium containing D-glucose 0.5% andmFPxBA 0.1% and shake cultured at 30° C. at 125 strokes/min. After 45hours, the cells were recovered by centrifugation, and were re-suspendedin 200 mL of M9 medium containing D-glucose 0.5% and mFPxBA 0.1% but notcontaining inorganic nitrogen source (NH₄Cl), then further shakecultured at 30° C. in 125 strokes/min. After 47 hours, cells wererecovered by centrifugation, washed once with cold methanol andlyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 57, result of identification, and weight obtained and yield of thelyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PH A, which contains 3HmFPxB as a monomer unit. Theextracted PHA mainly consists of 3HmFPxB unit as a main component, andis considered to be a mixture containing more than one of compoundsselected from the group consisting of 3-hydroxybutyric acid,3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid,and 3-hydroxydodecanoic acid, as a monomer unit.

Example 50

Pseudomonas cichorii H45 was inoculated in 200 ml of M9 mediumcontaining D-glucose 0.5% and mFPxBA 0.1% and was cultured at 30° C.with shaking at 125 strokes/min. After 96 hours, cultured cells wererecovered by centrifugation, and re-suspended in M9 medium containingD-glucose 0.5% and mFPxBA 0.1% but not containing the inorganic nitrogensource NH₄Cl, then further shake cultured at 30° C., 125 strokes/min.After 64 hours, cells were recovered by centrifugation, washed once withcold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 58, result of identification, and weight obtained and yield of thelyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA containing 3HmFPxB as a major monomer unit.

Example 51

Pseudomonas cichorii YN2 was inoculated in 200 mL of M9 mediumcontaining polypeptone 0.5% and mFPxBA 0.1% and was cultured at 30° C.with shaking at 125 strokes/min. After 24 hours, cultured cells wererecovered by centrifugation, and re-suspended in 200 ml of M9 mediumcontaining sodium pyruvate 0.5% and mFPxBA 0.1% but not containing theinorganic nitrogen source NH₄Cl, then further shake cultured at 30° C.,125 strokes/min. After 24 hours, bacterial cells were recovered bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 59, result of identification, and weight obtained and yield of thelyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA containing 3HmFPxB as a major monomer unit.

Example 52

Pseudomonas cichorii H45 was inoculated in 200 mL of M9 mediumcontaining polypeptone 0.5% and mFPxBA 0.1% and was cultured at 30° C.with shaking at 125 strokes/min. After 24 hours, cultured cells wererecovered by centrifugation, and were re-suspended in 200 mL of M9medium containing sodium pyruvate 0.5% and mFPxBA 0.1% but notcontaining inorganic nitrogen source NH₄Cl, then further shake culturedat 30° C., 125 strokes/min. After 24 hours, the cells were recovered bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 20 mL, and stirred at60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

The thus obtained PHA was subjected to methanolysis, and the product wasanalyzed using gas-chromatography mass spectrograph (GC-MS, ShimadzuQP-5050, EI method) to identify methyl esterified PHA monomer units. InTable 60, result of identification, and weight obtained and yield of thelyophilized pellet and the recovered polymer are shown. The thusobtained PHA is the PHA containing 3HmFPxB as a major monomer unit.

Example 53

<Production of PHA Containing HFPxV Unit by Strain YN2 (One Step cultureUsing Polypeptone)>

The strain YN2 was inoculated in 200 mL of M9 medium containingpolypeptone (Wako Pure Chemicals Co.) 0.5% and5-(4-fluorophenoxy)valeric acid (FPxVA) 0.1% and was cultured at 30° C.with shaking at 125 strokes/min. After 27 hours, cells were recovered bycentrifugation, washed once with cold methanol, lyophilized and wereweighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 20 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED—C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, molecularweight and analytical result of the monomer unit are shown in Table 61.Ratio of monomer unit was calculated by area ratio of GC-MS total ionchromatogram (TIC). Mass spectra obtained by GC-MS of 3-hydroxybutyricacid methyl ester and 3-hydroxy-5-(4-fluorophenoxy)valeric acid methylester are shown in FIG. 24 and FIG. 25.

Result indicates that PHA copolymer containing3-hydroxy-5-(4-fluorophenoxy)valeric acid unit can be produced by thestrain YN2 with a substrate 5-(4-fluorophenoxy)valeric acid.

Example 54

<Production of PHA Containing HFPV Unit by Strain YN2 (One Step CultureUsing Polypeptone)>

The strain YN2 was inoculated in 200 mL of M9 medium containingpolypeptone (Wako Pure Chemicals Co.) 0.5% and 5-(4-fluorophenyl)valericacid (FPVA) 0.1% and was cultured at 30° C. with shaking at 125strokes/min. After 27 hours, the cells were recovered by centrifugation,washed once with cold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 20 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, molecularweight and analytical result of the monomer unit are shown in Table 62.Ratio of monomer unit was calculated by area ratio of GC-MS total ionchromatogram (TIC). Mass spectra obtained by GC-MS of 3-hydroxybutyricacid methyl ester and 3-hydroxy-5-(4-fluorophenyl)valeric acid methylester are shown in FIG. 26 and FIG. 27.

Result indicates that PHA copolymer containing3-hydroxy-5-(4-fluorophenyl)valeric acid unit can be produced by thestrain YN2 with a substrate 5-(4-fluorophenyl)valeric acid.

Example 55

<Production of PHA Containing HFPxV Unit by Strain YN2 (Two Step CultureUsing Glucose)>

The strain YN2 was inoculated in 200 mL of M9 medium containing glucose0.5% and 5-(4-fluorophenoxy) valeric acid (FPxVA) 0.1% and was culturedat 30° C. with shaking at 125 strokes/min. After 24 hours, the culturedcells were recovered by centrifugation, and were re-suspended in 200 mLof M9 medium containing glucose 0.5% and FPxBA 0.1% but not containingnitrogen source (NH₄Cl), then further shake cultured at 30° C., 125strokes/min. After 62 hours, the cells were recovered by centrifugation,washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 20 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 63. Mass spectra obtainedby GC-MS of. 3-hydroxyoctanoic acid methyl ester, 3-hydroxydecanoic acidmethyl ester and 3-hydroxy-5-(4-fluorophenoxy)valeric acid (3HFPxV)methyl ester obtained by GC-MS measurement are shown in FIG. 28 to FIG.30.

Result indicates that PHA copolymer containing3-hydroxy-5-(4-fluorophenoxy)valeric acid (3HFPxV) unit can be producedby the strain YN2 with a substrate 5-(4-fluorophenoxy)valeric acid.

Example 56

<Production of PHA Containing HFPV Unit by Strain YN2 (Two Step CultureUsing Glucose)>

The strain YN2 was inoculated in 200 mL of M9 medium containing glucose0.5% and 5-(4-fluorophenyl) valeric acid (FPVA) 0.1% and was cultured at30° C. with shaking at 125 strokes/min. After 24 hours, the culturedcells were recovered by centrifugation, and were re-suspended in 200 mLof M9 medium containing glucose 0.5% and FPxVA 0.1% but not containingthe nitrogen source (NH₄Cl), then further shake cultured at 30° C. in125 strokes/min. After 62 hours, the cells were recovered bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 20 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The,mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 64. Mass spectra obtainedby GC-MS of 3-hydroxyoctanoic acid methyl ester, 3-hydroxydecanoic acidmethyl ester and 3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) methylester obtained by GC-MS measurement are shown in FIG. 31 to FIG. 33.

Result indicates that PHA copolymer containing3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) unit can be produced bythe strain YN2 with a substrate 5-(4-fluorophenyl)valeric acid.

Example 57

<Production of PHA Containing HFPV Unit and HFPxV Unit by Strain YN2(Two Step Culture Using Glucose)>

Two M9 media containing glucose 0.5% and 5-(4-fluorophenyl)valeric acid(FPVA) 0.1%, and containing glucose 0.5% and 5-(4-fluorophenoxy)valericacid (FPxVA) 0.1% were prepared respectively. Cells of strain YN2 wereinoculated in 200 mL of each M9 medium, and cultured at 30° C. withshaking at 125 strokes/min. After 94 hours, the cultured cells wererecovered by centrifugation, and the cells of these two cultures werere-suspended together in 200 mL of M9 medium containing glucose 0.5%,FPVA 0.1% and FPxVA 0.1% but not containing nitrogen source (NH₄Cl),then further shake cultured at 30° C., 125 strokes/min. After 24 hours,the cells were recovered by centrifugation, washed once with coldmethanol and lyophilized.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 20 hours to extract PHA. The extract was filtered usingmembrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain PHA by vacuum drying.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 65. Mass spectra obtainedby GC-MS of 3-hydroxyoctanoic acid methyl ester, 3-hydroxydecanoic acidmethyl ester, 3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) methylester and 3-hydroxy-5-(4-fluorophenoxy)valeric acid (3HFPxV) methylester obtained by GC-MS measurement are shown in FIG. 34 to FIG. 37.

Results indicate that PHA copolymer containing3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV) unit and3-hydroxy-5-(4-fluorophenoxy)valeric acid (3HFPxV) unit can be producedby the strain YN2 with a substrate 5-(4-fluorophenyl)valeric acid and5-(4-fluorophenoxy) valeric acid.

Example 58

<Production of PHA Containing HPxN Unit, HPxHp Unit and HPxV Unit byStrain YN2 (One Step Culture Using Polypeptone)>

The strain YN2 was inoculated in 200 mL of M9 medium containingpolypeptone (Wako Pure Chemicals Co.) 0.5% and 11-phenoxyundecanoic acid(PxUDA) 0.1% and was cultured at 30° C. with shaking 125 strokes/min.After 64 hours, the cells were recovered by centrifugation, washed oncewith cold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in acetone 100 mL, and stirred atroom temperature (23° C.) for 72 hours to extract polymer. The extractwas filtered using membrane filter, pore size 0.45 μm, concentratedusing rotary evaporator, precipitated the concentrate with coldmethanol, and recovered the precipitate to obtain the polymer by vacuumdrying, then weighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 66. Mass spectra obtainedby GC-MS of 3-hydroxybutyric acid methyl ester, 3-hydroxyoctanoic acidmethyl ester, 3-hydroxydecanoic acid methyl ester,3-hydroxy-5-phenoxyvaleric acid (3HPxV) methyl ester,3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) methyl ester and3-hydroxy-9-phenoxynonanoic acid (3HPxN) methyl ester are shown in FIG.38 to FIG. 43.

Result indicates that PHA copolymer containing three units of3-hydroxy-5-phenoxy valeric acid (3HPxV), 3-hydroxy-7-phenoxyheptanoicacid (3HPxHp) and 3-hydroxy-9-phenoxynonanoic acid (3HPxN) can beproduced by strain YN2 with a substrate 11-phenoxyundecanoic acid.

Example 59

<Production of PHA Containing HPxN Unit, HPxHp Unit and HPxV Unit byStrain YN2 (Two Steps Culture Using Glucose)>

The strain YN2 was inoculated in 200 mL of M9 medium containing glucose0.5% and 11-phenoxyundecanoic acid (PxUDA) 0.1% and was cultured at 30°C. with shaking at 125 strokes/min. After 64 hours, the cultured cellswere recovered by centrifugation, and were re-suspended in woo mL of M9medium 200 mL containing glucose 0.5% and PxUDA 0.1% but not containingnitrogen source (NH₄Cl), then further shake cultured at 30° C., 125strokes/min. After 24 hours, the cells were recovered by centrifugation,washed once with cold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 24 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 67. Mass spectra obtainedby GC-MS of 3-hydroxybutyric acid methyl ester, 3-hydroxyhexanoic acidmethyl ester, 3-hydroxyoctanoic acid methyl ester, 3-hydroxydecanoicacid methyl ester, 3-hydroxydodecanoic acid methyl ester,3-hydroxydodecenoic acid methyl ester, 3-hydroxy-5-phenoxyvaleric acid(3HPxV) methyl ester, 3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) methylester and 3-hydroxy-9-phenoxynonanoic acid (3HPxN) methyl ester areshown in FIG. 44 to FIG. 52.

Result indicates that PHA copolymer containing three units of3-hydroxy-5-phenoxyvaleric acid (3HPxV), 3-hydroxy-7-phenoxyheptanoicacid (3HPxHp) and 3-hydroxy-9-phenoxynonanoic acid (3HPxN) can beproduced by the strain YN2 with a substrate 11-phenoxyundecanoic acid.

Example 60

<Production of PHA Containing HPxN Unit, HPxHp Unit and HPxV Unit byStrain H45 (Two Steps Culture Using Glucose)>

Strain H45 was inoculated in 200 mL of M9 medium containing glucose 0.5%and 11-phenoxyundecanoic acid (PxUDA) 0.1% and was cultured at 30° C.with shaking at 125 strokes/min. After 64 hours, the cultured cells wererecovered by centrifugation, and were re-suspended in 200 mL of M9medium containing glucose 0.5% and PxUDA 0.1% but not containingnitrogen source (NH₄Cl), then further shake cultured at 30° C., 125strokes/min. After 24 hours, the cells were recovered by centrifugation,washed once with cold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 24 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 68. Mass spectra obtainedby GC-MS of 3-hydroxybutyric acid methyl ester, 3-hydroxyhexanoic acidmethyl ester, 3-hydroxyoctanoic acid methyl ester, 3-hydroxydecanoicacid methyl ester, 3-hydroxydodecanoic acid methyl ester,3-hydroxydodecenoic acid methyl ester, 3-hydroxy-5-phenoxyvaleric acid(3HPxV) methyl ester, 3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) methylester and 3-hydroxy-9-phenoxynonanoic acid (3HPxN) methyl ester areshown in FIG. 53 to FIG. 61.

Result indicates that PHA copolymer containing three units of3-hydroxy-5-phenoxyvaleric acid (3HPxV), 3-hydroxy-7-phenoxyheptanoicacid (3HPxHp) and 3-hydroxy-9-phenoxynonanoic acid (3HPxN) can beproduced by the strain H45 with a substrate 11-phenoxyundecanoic acid.

Example 61

<Production of PHA Containing HPxO Unit, HPxHx Unit and HPxB Unit byStrain YN2 (One Step Culture Using Polypeptone)>

The strain YN2 was inoculated in 200 mL of M9 medium containingpolypeptone (Wako Pure Chemicals—Co.) 0.5% and 8-phenoxyoctanoic acid(PxOA) 0.1% and was shake cultured at 30° C. in 125 strokes/min. After24 hours, the cells were recovered by centrifugation, washed once withcold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 24 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 69. Mass spectra obtainedby GC-MS of 3-hydroxybutyric acid methyl ester,3-hydroxy-4-phenoxybutyric acid (3HPxB) methyl ester,3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) methyl ester and3-hydroxy-8-phenoxyoctanoic acid (3HPxO) methyl ester are shown in FIG.62 to FIG. 65.

Result indicates that PHA copolymer containing three units of3-hydroxy-4-phenoxybutyric acid (3HPxB), 3-hydroxy-6-phenoxyhexanoicacid (3HPxHx) and 3-hydroxy-8-phenoxyoctanoic acid (3HPxO) can beproduced by the strain YN2 with a substrate 8-phenoxyoctanoic acid.

Example 62

<Production of PHA Containing HPxO Unit, HPxHx Unit and HPxB Unit byStrain H45 (One Step Culture Using Polypeptone)>

The strain H45 was inoculated in 200 mL of M9 medium containingpolypeptone (Wako Pure Chemicals Co.) 0.5% and 8-phenoxyoctanoic acid(PxOA) 0.1% and was shake cultured at 30° C. in 125 strokes/min. After24 hours, the cells were recovered by centrifugation, washed once withcold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 24 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 70. Mass spectra obtainedby GC-MS of 3-hydroxybutyric acid methyl ester,3-hydroxy-4-phenoxybutyric acid (3HPxB) methyl ester,3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) methyl ester and3-hydroxy-8-phenoxyoctanoic acid (3HPxO) methyl ester are shown in FIG.66 to FIG. 69.

Result indicates that PHA copolymer containing three units of3-hydroxy-4-phenoxybutyric acid (3HPxB), 3-hydroxy-6-phenoxyhexanoicacid (3HPxHx) and 3-hydroxy-8-phenoxyoctanoic acid (3HPxO) can beproduced by the strain H45 with a substrate 8-phenoxyoctanoic acid.

Example 63

<Production of PHA Containing HPxO Unit, HPxHx Unit and HPxB Unit byStrain YN2 (Two Steps Culture Using Glucose)>

The strain YN2 was inoculated in 200 mL of M9 medium containing glucose0.5% and 8-phenoxyoctanoic acid (PxOA) 0.1% and was cultured at 30° C.with shaking at 125 strokes/min. After 48 hours, the cultured cells wererecovered by centrifugation, and were re-suspended in 200 mL of M9medium containing glucose 0.5% and PxOA 0.1% but not containing nitrogensource (NH₄Cl), then further shake cultured at 30° C. in 125strokes/min. After 24 hours, bacterial cells were recovered bycentrifugation, washed once with cold methanol, lyophilized and wereweighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 24 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 71. Mass spectra obtainedby GC-MS measurement of 3-hydroxybutyric acid methyl ester,3-hydroxyoctanoic acid methyl ester, 3-hydroxydecanoic acid methylester, 3-hydroxy-4-phenoxybutyric acid (3HPxB) methyl ester,3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) methyl ester and3-hydroxy-8-phenoxyoctanoic acid (3HPxO) methyl ester are shown in FIG.70 to FIG. 75.

Result indicates that PHA copolymer containing three units of3-hydroxy-4-phenoxybutyric acid (3HPxB), 3-hydroxy-6-phenoxyhexanoicacid (3HPxHx) and 3-hydroxy-8-phenoxyoctanoic acid (3HPxO) can beproduced by the strain YN2 with a substrate 8-phenoxyoctanoic acid.

Example 64

<Production of PHA Containing HPxO Unit, HPxHx Unit and HPxB Unit byStrain H45 (Two Steps Culture Using Glucose)>

The strain H45 was inoculated in 200 mL of M9 medium containing glucose0.5% and 8-phenoxyoctanoic acid (PxOA) 0.1% and cultured at 30° C. withshaking at 125 strokes/min. After 48 hours, cultured bacterial cellswere recovered by centrifugation, and were re-suspended in 200 mL of M9medium containing glucose 0.5% and PxOA 0.1% but not containing nitrogensource (NH₄Cl), then further shake cultured at 30° C., 125 strokes/min.After 24 hours, the cells were recovered by centrifugation, washed oncewith cold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 24 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 72. Mass spectra obtainedby GC-MS measurement of 3-hydroxybutyric acid methyl ester,3-hydroxyhexanoic acid methyl ester, 3-hydroxyoctanoic acid methylester, 3-hydroxydecanoic acid methyl ester, 3-hydroxydodecanoic acidmethyl ester, 3-hydroxydodecenoic acid methyl ester,3-hydroxy-4-phenoxybutyric acid (3HPxB) methyl ester,3-hydroxy-6-phenoxyhexanoic acid (3HPxHx) methyl ester and3-hydroxy-8-phenoxyoctanoic acid (3HPxO) methyl ester are shown in FIG.76 to FIG. 84.

Result indicates that PHA copolymer containing three units of3-hydroxy-4-phenoxybutyric acid (3HPxB), 3-hydroxy-6-phenoxyhexanoicacid (3HPxHx) and 3-hydroxy-8-phenoxyoctanoic acid (3HPxO) can beproduced by the strain H45 with a substrate 8-phenoxyoctanoic acid.

Example 65

<Production of PHA Containing HPxHp Unit and HPxV Unit by Strain YN2(One Step culture Using Polypeptone)>

The strain YN2 was inoculated in 200 mL of M9 medium containingpolypeptone (Wako Pure Chemicals Co.) 0.5% and 7-phenoxyheptanoic acid(PxHpA) 0.1% and was cultured at 30° C. with shaking at 125 strokes/min.After 64 hours, the cells were recovered by centrifugation, washed oncewith cold methanol, lyophilized and weighed.

The lyophilized pellet was suspended in chloroform 100 mL, and stirredat 60° C. for 24 hours to extract polymer. The extract was filteredusing membrane filter, pore size 0.45 μm, concentrated using rotaryevaporator, precipitated the concentrate with cold methanol, andrecovered the precipitate to obtain the polymer by vacuum drying, thenweighed as such.

Molecular weight of the obtained polymer was measured by means ofgel-permeation chromatography (GPC: Toso, HLC-8020; Column: PolymerLaboratory, PL-gel, MIXED-C 5 μm; Solvent: chloroform; Molecular weight:reduced value for polystyrene).

Unit composition of the obtained polymer was analyzed by the followingmanner. The polymer sample 5 mg was poured into the 25 mL round-neckflask, and chloroform 2 mL and 2 mL of methanol containing sulfuric acid(3%, v/v). The mixture was refluxed at 100° C. for 3.5 hours, furtheradded water thereto for separation. The organic layer was analyzed bymeans of gas-chromatography mass spectrograph (GC-MS, Shimadzu QP-5050,Column: DB-WAXETR (J & W Inc.), EI method) to identify methyl esterifiedPHA monomer unit. Yields of bacterial cells and polymer, and analyticalresult of the monomer unit are shown in Table 73. Mass spectra obtainedby GC-MS of 3-hydroxybutyric acid methyl ester, 3-hydroxyoctanoic acidmethyl ester, 3-hydroxydecanoic acid methyl ester,3-hydroxy-5-phenoxyvaleric acid (3HPxV) methyl ester and3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) methyl ester are shown inFIG. 85 to FIG. 89.

Result indicates that PHA copolymer containing two units of3-hydroxy-5-phenoxyvaleric acid (3HPxV) and 3-hydroxy-7-phenoxyheptanoicacid (3HPxHp) can be produced by strain YN2 with a substrate7-phenoxyheptanoic acid.

Example 66

<Production of PHA Containing HPxHp Unit and HPxV Unit by Using strainH45 (Polypeptone, One-Step Culture)>

Strain H45 was inoculated in 200 mL of M9 medium containing 0.5%polypeptone (Wako Jun-Yaku Kogyo available) and 0.1% 7-phenoxyheptanoicacid (PxHpA) and cultured with shaking at 125 strokes/min at 30° C.After 64 hr, the cells were collected by centrifugation, washed oncewith cold methanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract the polymer. After filteredthrough a 0.45 μm pore-size membrane filter, the extract wasconcentrated by using a rotary evaporator and the concentrated solutionwas re-precipitated in cold methanol, and the precipitate was collected,vacuum-dried and weight as the polymer.

The molecular weight of the obtained polymer was measured by using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5 μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25 mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing 3% (v/v) sulfuric acid wasadded, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer wasanalyzed by a gas chromatograph—mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifya methyl esterified substance of the PHA monomer unit. Table 74 showsthe yield of the cells and polymers and the analyzed result of themonomer unit. Besides, FIGS. 90 to 92 show the mass spectra, obtained bythe GC-MS measurement of 3-hydroxybutyrate methyl ester,3-hydroxy-5-phenoxyvalerate (3HPxV) methyl ester and3-hydroxy-7-phenoxyheptanoate (3HPxHp) methyl ester, respectively.

From this result, it was revealed that PHA copolymer containing twounits of 3-hydroxy-5-phenoxyvaleric acid (3HPxV) and3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) could be produced usingstrain H45 and 7-phenoxyheptanoic acid as the substrate.

Example 67

<Production of PHA Containing HPxHp Unit and HPxV Unit by Using StrainYN2 (Glucose, Two-Step Culture)>

Strain YN2 was inoculated in 200 mL of M9 culture medium containing 0.5%glucose and 0.1% 7-phenoxyheptanoic acid (PxHpA) and cultured with 125strokes/min of shaking at 30° C. After 64 hr, the cells were collectedby centrifugation, re-suspended into 200 mL of M9 culture mediumcontaining 0.5% glucose and 0.1% PxHpA and no nitrogen source (NH₄Cl)and further cultured with 125 strokes/min of shaking at 30° C. After 24hr, the cells were collected by centrifugation, washed once with coldmethanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract polymer. After filtered through a0.45 μm pore-size membrane filter, the extract was concentrated by usinga rotary evaporator and the concentrated solution was re-precipitated incold methanol, and the precipitate was collected and vacuum-dried toobtain a polymer, then this polymer was weighed.

The molecular weight of the obtained polymer was measured by using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5 μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25 mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing 3% (v/v) sulfuric acid wasadded, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer. wasanalyzed on a gas chromatograph—mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifythe methyl esterified substance of the PHA monomer unit. Table 75 showsthe yield of the cells and polymer and the analyzed result of themonomer unit. Besides, FIGS. 93 to 100 show the mass spectra, obtainedby the GC-MS measurement of 3-hydroxybutyrate methyl ester,3-hydroxyhexanoate methyl ester, 3-hydroxyoctanoate methyl ester,3-hydroxydecanoate methyl ester, 3-hydroxydodecanoate methyl ester,3-hydroxydodecenoate methyl ester, 3-hydroxy-5-phenoxyvalerate (3HPxV)methyl ester and 3-hydroxy-7-phenoxyheptanoate (3HPxHp) methyl ester,respectively.

From this result, it was revealed that PHA copolymer containing twounits of 3-hydroxy-5-phenoxyvaleric acid (3HPxV) and3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) could be produced usingstrain YN2 and 7-phenoxyheptanoic acid as the substrate.

Example 68

<Production of PHA Containing HPxHp Unit and HPxV Unit by Using StrainH45 (Glucose, Two-Step Culture)>

Strain H45 was inoculated in 200 mL of M9 culture medium containing 0.5%glucose and 0.1% 7-phenoxyheptanoic acid (PxHpA) and cultured with 125strokes/min of shaking at 30° C. After 64 hr, the cells were collectedby centrifugation, re-suspended into 200 mL of M9 culture mediumcontaining 0.5% glucose and 0.1% PxHpA and no nitrogen source (NH₄Cl)and further cultured with 125 strokes/min of shaking at 30° C. After 24hr, the cells were collected by centrifugation, washed once with coldmethanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract polymer. After filtered through a0.45 μm pore-size membrane filter, the extract was concentrated using arotary evaporator and the concentrated solution was re-precipitated incold methanol, and the precipitate was collected and vacuum-dried toobtain a polymer, then this polymer was weighed.

The molecular weight of the obtained polymer was measured by using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5 μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25 mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing 3% (v/v) sulfuric acid wasadded, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer wasanalyzed on a gas chromatograph—mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifya methyl esterified substance of the PHA monomer unit. Table 76 showsthe yield of the cells and polymer and the analyzed result of themonomer unit. Besides, FIGS. 101 to 107 show the mass spectra, obtainedby the GC-MS measurement, of 3-hydroxyhexanoate methyl ester,3-hydroxyoctanoate methyl ester, 3-hydroxydecanoate methyl ester,3-hydroxydodecanoate methyl ester, 3-hydroxydodecenoate methyl ester,3-hydroxy-5-phenoxyvalerate (3HPxV) methyl ester and3-hydroxy-7-phenoxyheptanoate (3HPxHp) methyl ester, respectively.

From this result, it was revealed that PHA copolymer containing twounits of 3-hydroxy-5-phenoxyvaleric acid (3HPxV) and3-hydroxy-7-phenoxyheptanoic acid (3HPxHp) could be produced usingstrain H45 with 7-phenoxyheptanoic acid as the substrate.

Example 69

<Production of PHA Containing PHPxV Unit by Using Strain YN2 (SodiumMalate Two-Step Culture)>

Strain YN2 was inoculated in 200 mL of M9 culture medium containing 0.5%sodium malate and 0.1% 5-phenoxyvaleric acid (PxVA) and cultured with125 strokes/min of shaking at 30° C. After 60 hr, the cells werecollected by centrifugation, re-suspended into 200 mL of M9 culturemedium containing 0.5% sodium malate and 0.1% PxVA and no nitrogensource (NH₄Cl) and further cultured with 125 strokes/min of shaking at30° C. After 24 hr, the cells were collected by centrifugation, washedonce with cold methanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract polymer. After filtered through a0.45 μm pore-size membrane filter, the extract was concentrated by usinga rotary evaporator and the concentrated solution was re-precipitated incold methanol, further the precipitate alone was collected andvacuum-dried to obtain a polymer, then this polymer was weighed.

The molecular weight of the obtained polymer was measured by using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5 μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25 mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing 3% (v/v) sulfuric acid wasadded, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer wasanalyzed on a gas chromatograph—mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifya methyl esterified substance of the PHA monomer unit. Table 77 showsthe yield of the cells and polymer and the analyzed result of themonomer unit. Besides, FIGS. 108 to 114 show the mass spectra, obtainedby the GC-MS measurement, of 3-hydroxybutyrate methyl ester,3-hydroxyhexanoate methyl ester, 3-hydroxyoctanoate methyl ester,3-hydroxydecanoate methyl ester, 3-hydroxydodecanoate methyl ester,3-hydroxydodecenoate methyl ester and 3-hydroxy-5- phenoxyvalerate(3HPxV) methyl ester, respectively.

From this result, it was revealed that PHA copolymer containing3-hydroxy-5-phenoxyvaleric acid (3HPxV) unit could be produced usingstrain YN2 with 5-phenoxyvaleric acid as the substrate.

Example 70

<Production of PHA Containing HPxV Unit by Using Strain H45 (SodiumMalate Two-Step Culture)>

Strain H45 was inoculated in 200 mL of M9 culture containing 0.5% sodiummalate and 0.1% 5-phenoxyvaleric acid (PxVA) and cultured with 125strokes/min of shaking at 30° C. After 60 hr, the cells were collectedby centrifugation, re-suspended into 200 mL of M9 culture mediumcontaining 0.5% sodium maleate and 0.1% PxVA and no nitrogen source(NH₄Cl) and further cultured with 125 strokes/min of shaking at 30° C.After 24 hr, the cells were collected by centrifugation, washed oncewith cold methanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract polymer. After filtered through a0.45 μm pore-size membrane filter, the extract was concentrated using arotary evaporator and the concentrated solution was re-precipitated incold methanol, and the precipitate was collected and vacuum-dried toobtain a polymer, then this polymer was weighed.

The molecular weight of the obtained polymer was measured by using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25 mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing 3% (v/v) sulfuric acid wasadded, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer wasanalyzed on a gas chromatograph - mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifya methyl esterified substance of the PHA monomer unit. Table 78 showsthe yield of the cells and polymer and the analyzed result of themonomer unit. Besides, FIGS. 115 to 120 show the mass spectra, obtainedby the GC-MS measurement, of 3-hydroxyhexanoate methyl ester,3-hydroxyoctanoate methyl ester, 3-hydroxydecanoate methyl ester,3-hydroxydodecanoate methyl ester, 3-hydroxydodecenoate methyl ester and3-hydroxy-5-phenoxyvalerate (3HPxV) methyl ester, respectively.

From this result, it was revealed that PHA copolymer containing3-hydroxy-5-phenoxyvaleric acid (3HPxV) unit could be produced usingstrain H45 with 5-phenoxyvaleric acid as the substrate.

Example 71

<Production of PHA Containing HPV Unit by Using Strain YN2 (FructoseTwo-Step Culture)>

Strain YN2 was inoculated in 200 mL of M9 culture medium containing 0.5%fructose and 0.1% 5-phenylvaleric acid (PVA) and cultured with 125strokes/min of shaking at 30° C. After 120 hr, the cells were collectedby centrifugation, re-suspended into 200 mL of M9 culture mediumcontaining 0.5% fructose and 0.1% PxVA and no nitrogen source (NH₄Cl)and further cultured with 125 strokes/min of shaking at 30° C. After 50hr, the cells were collected by centrifugation, washed once with coldmethanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract polymer. After filtered through a0.45 μm pore-size membrane filter, the extract was concentrated by usinga rotary evaporator and the concentrated solution was re-precipitated incold methanol, further the precipitate alone was collected andvacuum-dried to obtain a polymer, then this polymer was weighed.

The molecular weight of the obtained polymer was measured using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5 μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25-mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing 3% (v/v) sulfuric acid wasadded, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer wasanalyzed on a gas chromatograph—mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifya methyl esterified substance of the PHA monomer unit. Table 79 showsthe yield of the cells and polymer and the analyzed result of themonomer unit. Besides, FIGS. 121 to 123 show the mass spectra, obtainedby the GC-MS measurement of 3-hydroxyoctanoate methyl ester,3-hydroxydecanoate methyl ester and 3-hydroxy-5-phenylvalerate (3HPV)methyl ester, respectively.

From this result, it was revealed that a PHA copolymer containing3-hydroxy-5-phenylvaleric acid (3HPV) unit could be produced usingstrain YN2 with 5-phenylvaleric acid as the substrate.

Example 72

<Production of PHA Containing HPV Unit by Using Strain YN2 (MannoseTwo-Step Culture)>

Strain YN2 was inoculated in 200 mL of M9 culture medium containing 0.5%mannose and 0.1% 5-phenylvaleric acid (PVA) and cultured with 125strokes/min of shaking at 30° C. After 43 hr, the cells were collectedby centrifugation, re-suspended into 200 mL of M9 culture mediumcontaining 0.5% mannose and 0.1% PxVA and no nitrogen source (NH₄Cl) andfurther cultured with 125 strokes/min of shaking at 30° C. After 91 hr,the cells were collected by centrifugation, washed once with coldmethanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract polymer. After filtered through a0.45 μm pore-size membrane filter, the extract was concentrated using arotary evaporator and the concentrated solution was re-precipitated incold methanol, and the precipitate was collected and vacuum-dried toobtain a polymer, then this polymer was weighed.

The molecular weight of the obtained polymer was measured by using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5 μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25 mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing. 3% (v/v) sulfuric acidwas added, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer wasanalyzed on a gas chromatograph - mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifya methyl esterified substance of the PHA monomer unit. Table 80 showsthe yield of the cells and polymer and the analyzed result of themonomer unit. Besides, FIGS. 124 and 125 show the mass spectra, obtainedby the GC-MS measurement, of 3-hydroxyoctanoate methyl ester and3-hydroxy-5-phenylvalerate (3HPV) methyl ester, respectively.

From this result, it was revealed that PHA copolymer containing3-hydroxy-5-phenylvaleric acid (3HPV) unit could be produced usingstrain YN2 with 5-phenylvaleric acid as the substrate.

Example 73

<Production of PHA Containing HPV Unit by Using Strain YN2 (SodiumLactate Two-Step Culture)>

Strain YN2 was inoculated in 200 mL of M9 culture medium containing 0.5%sodium lactate and 0.1% 5-phenylvaleric acid (PVA) and cultured with 125strokes/min of shaking at 30° C. After 46 hr, the cells were collectedby centrifugation, re-suspended into 200 mL of M9 culture containing0.5% sodium lactate and 0.1% PxVA and no nitrogen source (NH₄Cl) andfurther cultured with 125 strokes/min of shaking at 30° C. After 28 hr.the cells were collected by centrifugation, washed once with coldmethanol, lyophilized and weighed.

This lyophilized pellet was suspended in 100 mL of chloroform andstirred at 60° C. for 24 hr to extract polymer. After filtered through a0.45 μm pore-size membrane filter, the extract was concentrated by usinga rotary evaporator and the concentrated solution was re-precipitated incold methanol, and the precipitate was collected and vacuum-dried toobtain a polymer, then this polymer was weighed.

The molecular weight of the obtained polymer was measured using gelpermeation chromatography (GPC: Toso/HLC-8020; column: PolymerLaboratory/PL gel/MIXED-C/5 μm; solvent: chloroform; polystyrene reducedmolecular weight).

The unit composition of the obtained polymer was analyzed as follows: To5 mg of a polymer sample put in a 25 mL volume round bottom flask, 2 mLof chloroform and 2 mL of methanol containing 3% (v/v) sulfuric acid wasadded, the mixture was subjected to 100° C. and 3.5 hr reflux andseparated with a further addition of water, then the organic layer wasanalyzed on a gas chromatograph—mass spectrometer (GC-MS, ShimadzuQP-5050; column: DB-WAXETR (J & W Co. available); EI method) to identifya methyl esterified substance of the PHA monomer unit. Table 81 showsthe yield of the cells and polymer and the analyzed result of themonomer unit. Besides, FIGS. 126 to 129 show the mass spectra, obtainedby the GC-MS measurement, of 3-hydroxybutyrate methyl ester,3-hydroxyoctanoate methyl ester, 3-hydroxydecanoate methyl ester and3-hydroxy-5-phenylvalerate (3HPV) methyl ester, respectively.

From this result, it was revealed that PHA copolymer containing3-hydroxy-5-phenylvaleric acid (3HPV) unit could be produced usingstrain YN2 with 5-phenylvaleric acid as the substrate.

Example 74

<Production of PHA Containing HPxB Unit by Using Strain YN2 (DisodiumMalate Two-Step Culture)>

Pseudomonas cichorii strain YN2 was inoculated in 200 mL of 5 types ofM9 culture media each containing 0.1% 4-phenoxy-n-butyric acid (PxBA)and one of 0.5% of disodium malate semihydrate, L-sodium glutamatemonohydrate, D(+)-glucose and n-nonanoic acid and polypeptone (NihonSeiyaku) -respectively, and cultured with 125 strokes/min of shaking at30° C. After 48 hr, the cells were collected by centrifugation, washedonce with cold methanol and vacuum-dried.

These five pellets were suspended in 20 mL of chloroform separately andstirred at 60° C. for 20 hr to extract PHA. After filtered through a0.45 μm pore-size membrane filter, each extract was concentrated using arotary evaporator and the concentrated solution was re-precipitated incold methanol, and each precipitate was collected and vacuum-dried toobtain PHA. After subjected to methanolysis in accordance with the usualway, the obtained PHAs were analyzed using a gas chromatograph—massspectrometer (GC-MS, Shimadzu QP-5050; EI method) to identify the methylesterified substance of the PHA monomer unit. As a result, in case ofculturing with disodium malate as the growth carbon source, as shown inTable 82, PHA having a high proportion of 3-hydroxy-4-phenoxy-n-butyricacid (3HPxB) unit, a desired monomer unit derived from4-phenoxy-n-butyric acid, was obtained at a high yield. Furthermore,Table 83 shows the yield of the cells and polymer and the composition ofthe polymer in the case of culture using disodium malate.

Example 75

<Production of PHA Containing HPxB Unit by Using Strain YN2 (DisodiumMalate Two-Step Culture: Mass Culture)>

Pseudomonas cichorii strain YN2 was inoculated in 200 mL of M9 culturemedium containing 0.5% yeast extract (Oriental Yeast Industries), andcultured with 125 strokes/min of shaking at 30° C. as a seed culture. Ina 10 L jar fermenter containing 5 L of M9 medium containing 0.5% ofdisodium malate semihydrate and 0.1% 4-phenoxy-n-butyric acid, 50 mL ofseed cells was inoculated and shake cultured at 30° C. with 80stroke/min under aeration of 2.5 L/min. After 39 hr, the cells werecollected by centrifugation. This pellet was suspended in 120. mL of anapprox. 1.7% sodium hypochlorite solution and shaken at 4° C. for 2 hrto extract PHA. PHA was collected by centrifugation and dried to obtain56 mg of PHA per liter culture medium.

After subjected to methanolysis in accordance with the usual way, theobtained PHA was analyzed using a gas chromatograph—mass spectrometer(GC-MS, Shimadzu QP-5050; EI method) to identify a methyl esterifiedsubstance of the PHA monomer unit. As a result, the composition ratio(GC-MS, peak area ratio) of 3-hydroxy-4-phenoxy-n-butyric acid unit, adesired monomer unit derived from 4-phenoxy-n-butyric acid, was 99.7%.

TABLE 1 Weight of Weight of dry cell dry polymer Yield Carbon Source(alkanoate) (mg/L) (mg/L) (%) 6-phenoxyhexanoic acid 950 100 10.58-phenoxyoctanoic acid 820 90 11 11-phenoxyundecanoic acid 150 15 10

TABLE 2 NA:CHBA CDW PDW Yield Unit 5:5 756.0 89.1 11.8 NA, CHBA 1:9132.8 19.3 14.5 NA, CHBA CDW: Cell (Dry Weight) PDW: Polymer (DryWeight) Yield: PDW/CDW (%)

TABLE 3 Chemical Shift/ppm type Assignment 1.67 m c, d 2.39 t b 2.62 t e6.97 t h, j 7.12 t g, k 10.7 broad COOH

TABLE 4 P. cichorii strain H45 Cell (Dry weight) 750 mg/L Polymer (Dryweight) 400 mg/L Polymer (Dry weight)/Cell (Dry weight) 53%  MonomerUnit Composition (area ratio) 3-hydroxybutyric acid 0% 3-hydroxyvalericacid 0% 3-hydroxyhexanoic acid 0% 3-hydroxyheptanoic acid 13% 3-hydroxyoctanoic acid 0% 3-hydroxyoctanoic acid 3% 3-hydroxynonanoicacid 37%  3-hydroxydecanoic acid 0% 3-hydroxy-5-(4-fluorophenyl)valericacid 47% 

TABLE 5 P. cichorii strain YN2 Cell (Dry weight) 850 mg/L Polymer (Dryweight) 420 mg/L Polymer (Dry weight)/Cell (Dry weight) 49%  MonomerUnit Composition (area ratio) 3-hydroxybutyric acid 1% 3-hydroxyvalericacid 1% 3-hydroxyhexanoic acid 0% 3-hydroxyheptanoic acid 15% 3-hydroxyoctanoic acid 2% 3-hydroxynonanoic acid 68%  3-hydroxydecanoicacid 0% 3-hydroxy-5-(4-fluorophenyl)valeric acid 13% 

TABLE 6 P. putida P 91 strain Cell (Dry weight) 670 mg/L Polymer (Dryweight)  51 mg/L Polymer (Dry weight)/Cell (Dry weight) 8% Monomer UnitComposition (area ratio) 3-hydroxybutyric acid 0% 3-hydroxyvaleric acid1% 3-hydroxyhexanoic acid 0% 3-hydroxyheptanoic acid 11% 3-hydroxyoctanoic acid 2% 3-hydroxynonanoic acid 34%  3-hydroxydecanoicacid 0% 3-hydroxy-5-(4-fluorophenyl)valeric acid 52% 

TABLE 7 P. jessenii strain P161 Cell (Dry weight) 1200 mg/L Polymer (Dryweight)  640 mg/L Polymer (Dry weight)/Cell (Dry weight) 53%  MonomerUnit Composition (area ratio) 3-hydroxybutyric acid 1% 3-hydroxyvalericacid 1% 3-hydroxyhexanoic acid 0% 3-hydroxyheptanoic acid 17% 3-hydroxyoctanoic acid 3% 3-hydroxynonanoic acid 45%  3-hydroxydecanoicacid 0% 3-hydroxy-5-(4-fluorophenyl)valeric acid 33% 

TABLE 8 Purified PHA Monomer Unit Composition (area ratio)3-hydroxybutyric acid 0% 3-hydroxyvaleric acid 0% 3-hydroxyhexanoic acid0% 3-hydroxyheptanoic acid 0% 3-hydroxyoctanoic acid 0%3-hydroxynonanoic acid 0% 3-hydroxydecanoic acid 0%3-hydroxy-5-(4-fluorophenyl)valeric acid 100% 

TABLE 9 (Results of ¹H spectrometry) Resonance frequency: 400 MHz δ(ppm) Assignment 0.9 to 1.7 broad peak → impurity 1.8 to 1.9 m: 2H, —CH₂→ d 2.4 to 2.6 m: 4H, —CH₂ × 2 → b, e 5.2 to 5.3 m: 1H, —OCH → c 6.9 to7.0 t: 2H, proton at the ortho position of the F group → h, j 7.1 t: 2H,proton at the para position of the F group → g, k 7.3 s: solvent (CDCl₃)m: multiplet, t: triplet, s: singlet

TABLE 10 (Results of ¹³C spectrometry Resonance frequency: 100 MHz δ(ppm) Assignment 31.0 —CH₂ → d 35.9 —CH₂ → e 39.4 —CH₂ → b 70.5 —CH → c77.1 to 77.7 Solvent (CDCl₃) 115.5, 115.7 —CH at the ortho position ofthe F group → h, j 130.0 —CH at the meta position of the F group → g, k136.8 C at the para position of the F group → f 160.5, 163.0 —C at the Fsubstituent position → i 169.6 carbonyl group —C═O → a

TABLE 11 (Results of ¹H-NMR spectrum identification (see FIG. 4))Chemical Shift Integral /ppm /H type Identification 1.85 4 m c, d 2.46 2t b 3.95 2 t e 6.83 2 m h, j 6.97 2 t g, k 10.15  broad OH m: multiple,t: triplet, d: doublet

TABLE 12 (Various microorganisms and the yields of produced PHA)CDW(mg/L) PDW(mg/L) PDW/CDW(%) strain P91 650 50 7.7 (Example 8) strainYN2 1 1250 755 60.4 (Example 9) strain P161 1150 680 59.1 (Example 10)strain H45 1150 600 52.2 (Example 11) strain YN2 2 500 240 48.0 (Example12)

TABLE 13 (Molecular weight of PHA produced by each microorganism) Mn (×10⁴⁾ Mw (× 10⁵⁾ Mw/Mn strain P91 5.1 1.0 2.0 (Example 8) strain YN2 18.8 2.4 2.7 (Example 9) strain P161 6.8 1.8 2.7 (Example 10) strain H458.8 2.2 2.5 (Example 11) strain YN2 2 5.7 1.4 2.5 (Example 12)

TABLE 14 Dried cell Dried polymer Yield (mg/L) (mg/L) (polymer/cell, %)850 110 12.9

TABLE 15 3-hydroxyvaleric acid  1.4% 3-hydroxyheptanoic acid 29.3%3-hydroxyoctanoic acid  3.2% 3-hydroxynonanoic acid 64.6%3-hydroxy-5-(4-trifluomethylphenyl)  1.5% valeric acid

TABLE 16 Dried cell Dried polymer Yield (mg/L) (mg/L) (polymer/cell, %)720 29 4.0

TABLE 17 3-hydroxyvaleric acid 0.6% 3-hydroxyheptanoic acid 21.5% 3-hydroxyoctanoic acid 4.0% 3-hydroxynonanoic acid 70.5% 3-hydroxydecanoic acid 1.1% 3-hydroxy-5-(4-trifluomethylphenyl) 2.3%valeric acid

TABLE 18 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 1300 1000 Polymer (Dry Weight) (mg/L)  945  570 Polymer(Dry Weight) / 73%  57%  Cell (Dry Weight) Monomer Unit Composition(area ratio) 3-hydroxybutyric acid 0% 1% 3-hydroxyvaleric acid 0% 1%3-hydroxyhexanoic acid 0% 0% 3-hydroxyheptanoic acid 0% 14% 3-hydroxyoctanoic acid 1% 2% 3-hydroxynonanoic acid 0% 70% 3-hydroxydecanoic acid 2% 0% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-5-phenylvaleric acid 97%  12% 

TABLE 19 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 750 800 Polymer (Dry Weight) (mg/L) 400 385 Polymer (DryWeight) / 53%  48%  Cell (Dry Weight) Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 0% 0% 3-hydroxyvaleric acid 0% 0%3-hydroxyhexanoic acid 0% 0% 3-hydroxyheptanoic acJ.d 0% 14% 3-hydroxyoctanoic acid 0% 0% 3-hydroxynonanoic acid 0% 76% 3-hydroxydecanoic acid 0% 0% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-5-phenylvaleric acid 100%  10% 

TABLE 20 Carbon Source for growth D-mannose D-fructose Cell (Dry Weight)(mg/L) 780 760 Polymer (Dry Weight) (mg/L) 452 418 Polymer (Dry Weight)/ 58%  55%  Cell (Dry Weight) Monomer Unit Composition (area ratio)3-hydroxybutyric acid 0% 0% 3-hydroxyvaleric acid 0% 0%3-hydroxyhexanoic acid 0% 0% 3-hydrdxyheptanoic acid 0% 0%3-hydroxyoctanoic acid 2% 1% 3-hydroxynonanoic acid 0% 0%3-hydroxydecanoic acid 0% 1% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-5-phenylvaleric acid 98%  98% 

TABLE 21 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 1150 1180 Polymer (Dry Weight) (mg/L)  830  752 Polymer(Dry Weight) / 72%  64%  Cell (Dry Weight) Monomer Unit Composition(area ratio) 3-hydroxybutyric acid 0% 2% 3-hydroxyvaleric acid 0% 1%3-hydroxyhexanoic acid 0% 0% 3-hydroxyheptanoic acid 0% 17% 3-hydroxyoctanoic acid 1% 3% 3-hydroxynonanoic acid 0% 44% 3-hydroxydecanoic acid 3% 0% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-5-phenylvaleric acid 96%  33% 

TABLE 22 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 1250 920 Polymer (Dry Weight) (mg/L)  900 543 Polymer(Dry Weight) / 72%  59%  Cell (Dry Weight) Monomer Unit Composition(area ratio) 3-hydroxybutyric acid 0% 0% 3-hydroxyvaleric acid 0% 0%3-hydroxyhexanoic acid 0% 0% 3-hydroxyheptanoic acid 0% 11% 3-hydroxyoctanoic acid 1% 0% 3-hydroxynonanoic acid 0% 80% 3-hydroxydecanoic acid 2% 0% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-5-(4-fluorophenyl)- 97%  9%valeric acid

TABLE 23 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 1100 900 Polymer (Dry Weight) (mg/L)  143 119 Polymer(Dry Weight) / 13%  13%  Cell (Dry Weight) Monomer Unit Composition(area ratio) 3-hydroxybutyric acid 2% 2% 3-hydroxyvaleric acid 0% 1%3-hydroxyhexanoic acid 1% 0% 3-hydroxyheptanoic acid 0% 18% 3-hydroxyoctanoic acid 1% 3% 3-hydroxynonanoic acid 0% 48% 3-hydroxydecanoic acid 0% 0% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-6-phenylhexanoic acid 96%  28% 

TABLE 24 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 685 440 Polymer (Dry Weight) (mg/L) 137 263 Polymer (DryWeight) / 20%  60%  Cell (Dry Weight) Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 0% 0% 3-hydroxyvaleric acid 0% 1%3-hydroxyhexanoic acid 0% 1% 3-hydroxyheptanoic acid 0% 30% 3-hydroxyoctanoic acid 3% 4% 3-hydroxynonanoic acid 0% 62% 3-hydroxydecanoic acid 4% 1% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 1% 1% 3-hydroxy-4-phenoxybutyric acid 92%  0%

TABLE 25 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 450 340 Polymer (Dry Weight) (mg/L)  18 216 Polymer (DryWeight) / 4% 64%  Cell (Dry Weight) Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 0% 0% 3-hydroxyvaleric acid 0% 1%3-hydroxyhexanoic acid 1% 0% 3-hydroxyheptanoic acid 0% 28% 3-hydroxyoctanoic acid 5% 4% 3-hydroxynonanoic acid 0% 67% 3-hydroxydecanoic acid 5% 0% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 1% 0% 3-hydroxy-4-phenoxybutyric acid 88%  0%

TABLE 26 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 600 400 Polymer (Dry Weight) (mg/L)  51 144 Polymer (DryWeight) / 9% 36%  Cell (Dry Weight) Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 3% 0% 3-hydroxyvaleric acid 0% 1%3-hydroxyhexanoic acid 1% 1% 3-hydroxyheptanoic acid 0% 26% 3-hydroxyoctanoic acid 9% 5% 3-hydroxynonanoic acid 0% 63% 3-hydroxydecanoic acid 11%  2% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-4-phenoxybutyric acid 76%  2%

TABLE 27 n-nonanoic Carbon Source for growth D-glucose acid Cell (DryWeight) (mg/L) 1150 900 Polymer (Dry Weight) (mg/L)  590 420 Polymer(Dry Weight) / 51%  47%  Cell (Dry Weight) Monomer Unit Composition(area ratio) 3-hydroxybutyric acid 1% 0% 3-hydroxyvaleric acid 0% 0%3-hydroxyhexanoic acid 0% 0% 3-hydroxyheptanoic acid 0% 13% 3-hydroxyoctanoic acid 0% 5% 3-hydroxynonanoic acid 0% 69% 3-hydroxydecanoic acid 10%  0% 3-hydroxyundecanoic acid 0% 0%3-hydroxydodecanoic acid 0% 0% 3-hydroxy-5-phenylvaleric acid 89%  13% 

TABLE 28 CDW (mg/L) PDW (mg/L) Yield (%) (1) YE 1225 488 39.8 (2) BE 600 185 30.8 (3) CA  950 445 46.8 (4) PP 1200 755 62.9 CDW: Cell (DryWeight) (mg/L) PDW: Polymer (Dry Weight) (mg/L) Yield: PDW/CDW (%)

TABLE 29 CDW (mg/L) PDW (mg/L) Yield (%) (1) YE 1100 225 20.5 (2) PP1200 850 70.8 CDW: Cell (Dry Weight) (mg/L) PDW: Polymer (Dry Weight)(mg/L) Yield: PDW/CDW (%)

TABLE 30 CDW (mg/L) PDW (mg/L) Yield (%) (1) YE 1050 205 19.5 (2) PP1000 345 34.5 CDW: Cell (Dry Weight) (mg/L) PDW: Polymer (Dry Weight)(mg/L) Yield: PDW/CDW (%)

TABLE 31 CDW (mg/L) PDW (mg/L) Yield (%) (1) YE 750 220 29.3 (2) SG 700260 37.1 (3) CA 900 340 37.7 (4) PP 1100  450 40.9 CDW: Cell (DryWeight) (mg/L) PDW: Polymer (Dry Weight) (mg/L) Yield: PDW/CDW (%)

TABLE 32 CDW (mg/L) PDW (mg/L) Yield (%) (1) YE 950 325 34.2 (2) SG 750240 32.0 (3) BE 450 130 28.9 (4) PP 1000  450 45.0 CDW: Cell (DryWeight) (mg/L) PDW: Polymer (Dry Weight) (mg/L) Yield: PDW/CDW (%)

TABLE 33 Identification results of the ¹H-NMR spectral patterns (of.FIG. 14) Chemical shift (ppm) Identification 2.80 b1 4.24 d1 5.50 c17.00 f1, j1 8.20 g1, l1

TABLE 34 Cell (Dry Weight) 280 mg/L Polymer (Dry Weight)  30 mg/LPolymer (Dry Weight) / Cell (Dry weight) 10.7% Ratio of monomer unitdetermined by NMR (mole ratio) 3HNO₂PxB contained in PHA 3.3 mol % Fattyacid-derived monomer unit composition (peak area ratio) 3-hydroxybutyricacid  9.3% 3-hydroxyhexanoic acid  2.0% 3-hydroxyoctanoic acid 12.2%3-hydroxydecanoic acid 47.9% 3-hydroxydodecanoic acid 15.7%3-hydroxydodecenoic acid 12.9%

TABLE 35 Cell (Dry Weight) 250 mg/L Polymer (Dry Weight)  30 mg/LPolymer (Dry Weight) / Cell (Dry Weight) 12.0% Ratio of monomer unitdetermined by NMR (mole ratio) 3HNO₂PxB contained in PHA 4.3 mol % Fattyacid-derived monomer unit composition (peak area ratio) 3-hydroxybutyricacid  1.3% 3-hydroxyhexanoic acid  0.0% 3-hydroxyoctanoic acid 12.6%3-hydroxydecanoic acid 38.8% 3-hydroxydodecanoic acid 22.7%3-hydroxydodecenoic acid 24.6%

TABLE 36 Cell (Dry Weight) 785 mg/L Polymer (Dry Weight)  55 mg/LPolymer (Dry Weight) / Cell (Dry Weight)  7.0% Ratio of monomer unitdetermined by NMR (mole ratio) 3HNO₂PxB contained in PHA 3.8 mol % Fattyacid-derived monomer unit composition (peak area ratio) 3-hydroxybutyricacid  2.9% 3-hydroxyhexanoic acid  1.5% 3-hydroxyoctanoic acid 12.13-hydroxydecanoic acid 40.0% 3-hydroxydodecanoic acid 14.7%3-hydroxydodecenoic acid 28.8%

TABLE 37 Identification results of ¹H-NMR spectral patterns (of. FIG.15) Chemical shift (ppm) Identification 2.79 b1 4.18 d1 5.51 c1 6.98 f1,j1 7.58 g1, l1

TABLE 38 PHA production by Pseudorionas cichorii YN2 Cell (Dry Weight)900 mg/L Polymer (Dry Weight) 180 mg/L Polymer (Dry Weight) / Cell (DryWeight) 20.0% Ratio of monomer unit determined by NMR (mole ratio)3HCNPxBA contained in PHA 4.1 mol % Fatty acid-derived monomer unitcomposition (peak area ratio) 3-hydroxybutyric acid  9.3%3-hydroxyhexanoic acid  2.0% 3-hydroxyoctanoic acid 12.2%3-hydroxydecanoic acid 47.9% 3-hydroxydodecanoic acid 15.7%3-hydroxydodecenoic acid 12.9%

TABLE 39 PHA production by Pseudomonas cichorii H45 Cell (Dry Weight)775 mg/L Polymer (Dry Weight) 150 mg/L Polymer (Dry Weight) / Cell (DryWeight) 19.4% Ratio of monomer unit determined by NMR (inoie ratio)3HCNPxBA contained in PHA 3.2 mol % Fatty acid-derived monomer unitcomposition (peak area ratio) 3-hydroxybutyric acid 70.5%3-hydroxyhexanoic acid  1.0% 3-hydroxyoctanoic acid 10.3%3-hydroxydecanoic acid 13.4% 3-hydroxydodecanoic acid  2.3%3-hydroxydodecenoic acid  2.6%

TABLE 40 PHA production by Pseudomonas cichorii YN2 Cell (Dry Weight)930 mg/L Polymer (Dry Weight) 200 mg/L Polymer (Dry Weight) / Cell (DryWeight) 21.5% Ratio of monomer unit determined by NMR (mole ratio)3HCNPxBA contained in PHA 4.5 mol % Fatty acid-derived monomer unitcomposition (peak area ratio) 3-hydroxybutyric acid 12.5%3-hydroxyhexanoic acid  2.0% 3-hydroxyoctanoic acid 12.2%3-hydroxydecanoic acid 47.3% 3-hydroxydodecanoic acid 15.2%3-hydroxydodecenoic acid 10.8%

TABLE 41 PHA production by Pseudomonas cichorii H45 Cell (Dry Weight)750 mg/L Polymer (Dry Weight) 135 mg/L Polymer (Dry Weight) / Cell (DryWeight) 18.0% Ratio of monomer unit determined by NMR (mole ratio)3RCNPxBA contained in PHA 4.4 mol % Fatty acid-derived monomer unitcomposition (peak area ratio) 3-hydroxybutyric acid 25.0%3-hydroxyhexanoic acid  2.1% 3-hydroxyoctanoic acid 21.4%3-hydroxydecanoic acid 37.3% 3-hydroxydodecanoic acid  6.0%3-hydroxydodecenoic acid  8.2%

TABLE 42 PHA production by Pseudomonas cichorii YN2 Cell (Dry Weight)1030 mg/L Polymer (Dry Weight)  130 mg/L Polymer (Dry Weight) / Cell(Dry Weight) 12.6% Ratio of monomer unit determined by NMR (mole ratio)3HCNPxBA contained in PHA 7.2 mol % Fatty acid-derived monomer unitcomposition (peak area ratio) 3-hydroxybutyric acid  7.3%3-hydroxyhexanoic acid  1.9% 3-hydroxyoctanoic acid 14.3%3-hydroxydecanoic acid 48.2% 3-hydroxydodecanoic acid 12.8%3-hydroxydodecenoic acid 15.5%

TABLE 43 PHA production by Pseudomonas cichorii H45 Cell (Dry Weight)695 mg/L Polymer (Dry Weight)  55 mg/L Polymer (Dry Weight) / Cell (DryWeight)  7.9% Ratio of monomer unit dE.termined by NMR (mole ratio)3HCNPxBA contained in PHA 2.6 mol % Fatty acid-derived monomer unitcomposition (peak area ratio) 3-hydroxybutyric acid  2.3%3-hydroxyhexanoic acid  1.7% 3-hydroxyoctanoic acid 19.5%3-hydroxydecanoic acid 52.1% 3-hydroxydodecanoic acid 10.3%3-hydroxydodecenoic acid 14.1%

TABLE 44 ¹H-NMR Spectrum (of. FIG. 16) Chemical shift Integral (ppm)value type Identification 2.11 2H quint CH₂ c 2.59 2H t CH₂ b 3.97 2H tCH₂ d 6.82 2H m g, i 6.95 2H m f, j 8.00 to 13.00 1H br OH

TABLE 45 ¹³C-NMR Spectrum (of. FIG. 17) Chemical shift (ppm) typeIdentification 24.21 s CH₂ c  30.39 s CH₂ b  66.96 s CH₂ d 115.23 &115.31 d f, j or g, i 115.56 & 115.79 d f, j or g, i 154.70 & 154.71 d e155.97 & 158.33 d h 179.40 s C═O a

TABLE 46 PHA production containing 3HpFPxB unit by strain YN2 Cell (DryWeight) 885 mg/L Polymer (Dry Weight) 220 mg/L Polymer (Dry Weight) /Cell (Dry Weight) 24.9%  Polymer Molecular Weight Mn = 42,400 Mw =90,600 Monomer Unit Composition (area ratio) 3-hydroxybutyric acid 1.8%3-hydroxyhexanoic acid 1.0% 3-hydroxyoctanoic acid 5.4% 3-hydroxydecanoic acid 10.4%  3-hydroxydodecanoic acid 2.9%3-hydroxydodecenoic acid 5.9% 3-hydroxy-4-(4-fluoro)phenoxy)butyric acid72.6% 

TABLE 47 ¹H-NMR Spectrum (of. FIG. 19) Chemical shift (ppm)Identification 2.76 2H, CH₂ b1 3.95 to 4.06 2H, CH₂ d1 5.46 1H, CH c16.71 to 6.90 4H, —C₆H_(4-  f1, g1, i1, j1)

TABLE 48 Production of PHA containing 3HpFPxB unit by culturing strainH45 Cell (dry weight) 640 mg/L Polymer (dry weight)  90 mg/L Polymer(dry weight)/Cell (dry weight) 14.0%  Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 4.6% 3-hydroxyhexanoic acid 2.0%3-hydroxyoctanoic acid 15.2%  3-hydroxydecanoic acid 19.8% 3-hydroxydodecanoic acid 4.0% 3-hydroxydodecenoic acid 7.4%3-hydroxy-4-(4-fluorophenoxy)butyric acid 47.0% 

TABLE 49 Production of PHA containing 3HpFPxB unit by culturing strainYN2 Cell (dry weight) 780 mg/L Polymer (dry weight) 200 mg/L Polymer(dry weight)/Cell (dry weight) 25.6%  Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 0.3% 3-hydroxyhexanoic acid 0.8%3-hydroxyoctanoic acid 3.9% 3-hydroxydecanoic acid 6.7%3-hydroxydodecanoic acid 2.1% 3-hydroxydodecenoic acicl 3.7%3-hydroxy-4-(4-fluorophenoxy)butyric acid 82.5% 

TABLE 50 Production of PHA containing 3HpFPXB unit by culturing strainH45 Cell (dry weight) 590 mg/L Polymer (dry weight)  45 mg/L Polymer(dry weight)/Cell (dry weight) 7.6% Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 0.2% 3-hydroxyhexanoic acid 1.9%3-hydroxyoctanoic acid 11.9%  3-hydroxydecanoic acid 12.1% 3-hydroxydodecanoic acid 2.2% 3-hydroxydodecenoic acid 5.0%3-hydroxy-4-(4-fluorophenoxy)butyric acid 66.7% 

TABLE 51 Production of PHA containing 3HpFPXB unit by culturing strainYN2 Cell (dry weight) 960 mg/L Polymer (dry weight) 155 mg/L Polymer(dry weight)/Cell (dry weight) 16.1%  Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 0.5% 3-hydroxyhexanoic acid 1.0%3-hydroxyoctanoic acid 8.9% 3-hydroxydecanoic acid 23.4% 3-hydroxydodecanoic acid 7.0% 3-hydroxydodecenoic acid 14.2% 3-hydroxy-4-(4-fluorophenoxy)butyric acid 45.0% 

TABLE 52 Production of PHA containing 3HpFPxB unit by culturing strainH45 Cell (dry weight) 545 mg/L Polymer (dry weight)  30 mg/L Polymer(dry weight)/Cell (dry weight) 5.5% Monomer Unit Composition (arearatio) 3-hydroxybutyric acid 2.3% 3-hydroxyhexanoic acid 1.6%3-hydroxyoctanoic acid 15.6%  3-hydroxydecanoic acid 36.9% 3-hydroxydodecanoic acid 8.7% 3-hydroxydodecenoic acid 12.9% 3-hydroxy-4-(4-fluorophenoxy)butyric acid 22.0% 

TABLE 53 ¹H-NMR Spectrum (of. FIG. 20) Chemical Integral shift (ppm)value type Identification 2.11 2H d, quint CH₂ c 2.59 2H t CH₂ b 3.97 2Ht CH₂ d 6.62 3H m h, i, j 7.21 1H m f 10.62  1H br OH

TABLE 54 ¹³C-NMR Spectrum (of. FIG. 21) Chemical shift (ppm) typeIdentification  24.1 s CH₂ c  30.34 s CH₂ b  66.62 s CH₂ d 101.23 &102.19 d f 107.39 & 107.60 d h 110.08 & 110.11 d j 130.04 & 130.14 d i159.92 & 160.03 d e 162.29 & 164.73 d g 179.19 s C═O a

TABLE 55 PHA production containing 3HmFPxB unit by strain YN2 Cell (DryWeight) 745 mg/L Polymer (Dry Weight)  80 mg/L Polymer (Dry Weight) /Cell (Dry Weight) 10.7%  Polymer Molecular Weight Mn = 34,500 Mw =75,200 Monomer Unit Composition (area ratio) 3-hydroxybutyric acid 1.2%3-hydroxyhexanoic acid 1.0% 3-hydroxyoctanoic acid 6.5%3-hydroxydecanoic acid 9.5% 3-hydroxydodecanoic acid 3.5%3-hydroxydodecenoic acid 5.9% 3-hydroxy-4-(3-fluorophenoxy)butyric acid72.5% 

TABLE 56 ¹H-NMR Spectrum (of. FIG. 23) Chemical shift (ppm)Identification 2.75 2H, CH₂ b1 4.00 2H, CH₂ d1 5.48 1H, CH c1 6.52 to6.62 3H, h1, i1, j1 7.26 3H, f1

TABLE 57 PHA production containing 3HmFPxB unit by strain H45 Cell (DryWeight) 630 mg/L Polymer (Dry Weight)  45 mg/L Polymer (Dry Weight) /Cell (Dry Weight) 7.1% Monomer Unit Composition (area ratio)3-hydroxybutyric acid 3.7% 3-hydroxyhexanoic acid 2.2% 3-hydroxyoctanoidacid 21.0%  3-hydroxydecanoic acid 29.2%  3-hydroxydodecanoic acid 8.2%3-hydroxydodecenoic acid 10.1%  3-hydroxy-4-(3-fluorophenoxy)butyricacid 25.6% 

TABLE 58 PHA production containing 3HmFPxB unit by strain H45 Cell (DryWeight) 515 mg/L Polymer (Dry Weight)  40 mg/L Polymer (Dry Weight) /Cell (Dry Weight) 7.8% Monomer Unit Composition (area ratio)3-hydroxybutyric acid 3.0% 3-hydroxyhexanoic acid 2.0% 3-hydroxyoctanoicacid 16.8%  3-hydroxydecanoic acid 16.8%  3-hydroxydodecanoic acid 4.7%3-hydroxydodecenoic acid 7.4% 3-hydroxy-4-(3-fluorophenoxy)butyric acid49.3% 

TABLE 59 PHA production containing 3HmFPxB unit by strain YN2 Cell (DryWeight) 900 mg/L Polymer (Dry Weight)  90 mg/L Polymer (Dry Weight) /Cell (Dry weight) 10.0%  Monomer Unit Composition (area ratio)3-hydroxybutyric acid 20.1%  3-hydroxyhexanoic acid 1.5%3-hydroxyoctanoic acid 9.8% 3-hydroxydecanoic acid 15.5% 3-hydroxydodecanoic acid 5.5% 3-hydroxydodecenoic acid 9.7%3-hydroxy-4-(3-fluorc)phenoxy)butyric acid 37.9% 

TABLE 60 PHA production containing 3HmFPxB unit by strain H45 Cell (DryWeight) 565 mg/L Polymer (Dry Weight)  25 mg/L Polymer (Dry Weight) /Cell (Dry Weight) 4.4% Monomer Unit Composition (area ratio)3-hydroxybutyric acid 4.2% 3-hydroxyhexanoic acid 1.9% 3-hydroxyoctanoicacid 17.8%  3-hydroxydecanoic acid 38.0%  3-hydroxydodecanoic acid 9.5%3-hydroxydodecenoic acid 13.8%  3-hydroxy-4-(3-fluorophenoxy) butyricacid 14.8% 

TABLE 61 Cell (Dry Weight) (mg/L) 665 Polymer (Dry Weight) (mg/L) 105Number Average Molecular Weight (Mn) × 10⁴ 1.6 Weight Average MolecularWeight (Mw) × 10⁴ 3.7 3-hydroxybutyric acid (%) 75.23-hydroxy-5-(4-fluorophenoxy)valeric acid (%) 24.8

TABLE 62 Cell (Dry Weight) (mg/L) 1120 Polymer (Dry Weight) (mg/L) 625Number Average Molecular Weight (Mn) × 10⁴ 4.8 Weight Average MolecularWeight (Mw) × 10⁴ 9.9 3-hydroxybutyric acid (%) 17.43-hydroxy-5-(4-fluorophenyl)valeric acid (%) 82.6

TABLE 63 Cell (Dry Weight) (mg/L) 835 Polymer (Dry weight) (mg/L) 395Number Average Molecular Weight (Mn) × 10⁴ 5.2 Weight Average MolecularWeight (Mw) × 10⁴ 14.9 3-hydroxyoctanoic acid (%) 10.6 3-hydroxydecanoicacid (%) 9.5 3-hydroxy-5-(4-fluorophenoxy)valeric acid (%) 79.9

TABLE 64 Cell (Dry Weight) (mg/L) 1450 Polymer (Dry Weight) (mg/L) 1010Number Average Molecular Weight (Mn) × 10⁴ 6.0 Weight Average MolecularWeight (Mw) × 10⁴ 14.8 3-hydroxyoctanoic acid (%) 2.8 3-hydroxydecanoicacid (%) 2.7 3-hydroxy-5-(4-fluorophenoxy)valeric acid (%) 94.5

TABLE 65 Cell (Dry Weight) (mg/L) 1605 Polymer (Dry Weight) (mg/L) 760Number Average Molecular Weight (Mn) × 10⁴ 4.6 Weight Average MolecularWeight (Mw) × 10⁴ 12.6 3-hydroxyoctanoic acid (%) 0.5 3-hydroxydecanoicacid (%) 0.4 3-hydroxy-5-(4-fluorophenyl)valeric acid (%) 90.03-hydroxy-5-(4-fluorophenoxy)valeric acid (%) 9.1

TABLE 66 Cell (Dry Weight) (mg/L) 1200 Polymer (Dry Weight) (mg/L) 500Number Average Molecular Weight (Mn) × 10⁴ 2.2 Weight Average MolecularWeight (Mw) × 10⁴ 4.9 3-hydroxybutyric acid (%) 3.4 3-hydroxyoctanoicacid (%) 0.3 3-hydroxydecanoic acid (%) 0.5 3-hydroxy-5-phenoxyvalericacid (%) 34.1 3-hydroxy-7-phenoxyheptanoic (%) 51.13-hydroxy-9-phenoxynonanoic acid (%) 10.6

TABLE 67 Cell (Dry Weight) (mg/L) 1305 Polymer (Dry Weight) (mg/L) 765Number Average Molecular Weight (Mn) × 10⁴ 5.0 Weight Average MolecularWeight (Mw) × 10⁴ 11.6 3-hydroxybutyric acid (%) 0.1 3-hydroxyhexanoicacid (%) 0.3 3-hydroxyoctanoic acid (%) 3.0 3-hydroxydecanoic acid (%)5.2 3-hydroxydodecanoic acid (%) 1.6 3-hydroxydodecenoic acid (%) 2.03-hydroxy-5-phenoxyvaleric acid (%) 40.7 3-hydroxy-7-phenoxyheptanoic(%) 40.4 3-hydroxy-9-phenoxynonanoic acid (%) 6.7

TABLE 68 Cell (Dry Weight) (mg/L) 1085 Polymer (Dry Weight) (mg/L) 585Number Average Molecular Weight (Mn) × 10⁴ 4.0 Weight Average MolecularWeight (Mw) × 10⁴ 8.9 3-hydroxybutyric acid (%) 0.4 3-hydroxyhexanoicacid (%) 0.3 3-hydroxyoctanoic acid (%) 3.2 3-hydroxydecanoic acid (%)5.1 3-hydroxydodecanoic acid (%) 1.1 3-hydroxydodecenoic acid (%) 0.93-hydroxy-5-phenoxyvaleric acid (%) 43.0 3-hydroxy-7-phenoxyheptanoic(%) 43.9 3-hydroxy-9-phenoxynonanoic acid (%) 2.1

TABLE 69 Cell (Dry Weight) (mg/L) 1100 Polymer (Dry Weight) (mg/L) 440Number Average Molecular Weight (Mn) × 10⁴ 4.0 Weight Average MolecularWeight (Mw) × 10⁴ 7.4 3-hydroxybutyric acid (%) 16.43-hydroxy-4-phenoxybutyric acid (%) 13.4 3-hydroxy-6-phenoxyhexanoicacid (%) 67.9 3-hydroxy-8-phenoxyoctanoic acid (%) 2.3

TABLE 70 Cell (Dry Weight) (mg/L) 860 Polymer (Dry Weight) (mg/L) 190Number Average Molecular Weight (Mn) × 10⁴ 3.4 Weight Average MolecularWeight (Mw) × 10⁴ 6.8 3-hydroxybutyric acid (%) 0.23-hydroxy-4-phenoxybutyric acid (%) 5.0 3-hydroxy-6-phenoxyhexanoic acid(%) 82.9 3-hydroxy-8-phenoxyoctanoic acid (%) 11.8

TABLE 71 Cell (Dry Weight) (mg/L) 1405 Polymer (Dry Weight) (mg/L) 700Number Average Molecular Weight (Mn) × 10⁴ 4.9 Weight Average MolecularWeight (Mw) × 10⁴ 10.7 3-hydroxybutyric acid (%) 4.8 3-hydroxyoctanoic(%) 1.2 3-hydroxydecanoic acid (%) 0.5 3-hydroxy-4-phenoxybutyric acid(%) 7.8 3-hydroxy-6-phenoxyhexanoic acid (%) 74.83-hydroxy-8-phenoxyoctanoic acid (%) 10.9

TABLE 77 Cell (Dry Weight) (mg/L) 1255 Polymer (Dry Weight) (mg/L) 560Number Average Molecular Weight (Mn) × 10⁴ 4.8 Weight Average MolecularWeight (Mw) × 10⁴ 9.7 3-hydroxybutyric acid (%) 0.2 3-hydroxyhexanoicacid (%) 0.1 3-hydroxyoctanoic acid (%) 0.9 3-hydroxydecanoic acid (%)0.9 3-hydroxydodecanoic acid (%) 0.9 3-hydroxydodecenoic acid (%) 0.23-hydroxy-4-phenoxybutyric acid (%) 2.5 3-hydroxy-6-phenoxyhexanoic acid(%) 82.8 3-hydroxy-8-phenoxyoctanoic acid (%) 12.3

TABLE 73 Cell (Dry Weight) (mg/L) 995 Polymer (Dry Weight) (mg/L) 505Number Average Molecular Weight (Mn) × 10⁴ 4.6 Weight Average MolecularWeight (Mw) × 10⁴ 9.2 3-hydroxybutyric acid (%) 1.4 3-hydroxyoctanoicacid (%) 0.1 3-hydroxydecanoic acid (%) 0.2 3-hydroxy-5-phenoxyvalericacid (%) 29.7 3-hydroxy-7-phenoxyheptanoic acid (%) 68.6

TABLE 74 Cell (Dry Weight) (mg/L) 815 Polymer (Dry Weight) (mg/L) 270Number Average Molecular Weight (Mn) × 10⁴ 3.3 Weight Average MolecularWeight (Mw) × 10⁴ 6.4 3-hydroxybutyric acid (%) 1.23-hydroxy-5-phenoxyvaleric acid (%) 26.7 3-hydroxy-7-phenoxyheptanoicacid (%) 72.1

TABLE 75 Cell (Dry Weight) (mg/L) 1520 Polymer (Dry Weight) (mg/L) 860Number Average Molecular Weight (Mn) × 10⁴ 6.1 Weight Average MolecularWeight (Mw) × 10⁴ 13.0 3-hydroxybutyric acid (%) 0.1 3-hydroxyhexanoicacid (%) 0.4 3-hydroxyoctanoic acid (%) 3.5 3-hydroxydecanoic acid (%)4.1 3-hydroxydodecanoic acid (%) 1.1 3-hydroxydodecenoic acid (%) 3.13-hydroxy-5-phenoxyvaleric acid (%) 55.0 3-hydroxy-7-phenoxyheptanoicacid (%) 32.7

TABLE 76 Cell (Dry Weight) (mg/L) 1305 Polymer (Dry Weight) (mg/L) 685Number Average Molecular Weight (Mn) × 10⁴ 4.1 Weight Average MolecularWeight (MW) × 10⁴ 8.8 3-hydroxyhexanoic acid (%) 0.1 3-hydroxyoctanoicacid (%) 1.3 3-hydroxydecanoic acid (%) 1.8 3-hydroxydodecanoic acid (%)0.4 3-hydroxydodecenoic acid (%) 0.6 3-hydroxy-5-phenoxyvaleric acid (%)36.1 3-hydroxy-7-phenoxyheptanoic acid (%) 59.7

TABLE 77 Cell (Dry Weight) (mg/L) 890 Polymer (Dry Weight) (mg/L) 420Number Average Molecular Weight (Mn) × 10⁴ 9.7 Weight Average MolecularWeight (Mw) × 10⁴ 29.7 3-hydroxybutyric acid (%) 0.2 3-hydroxyhexanoicacid (%) 0.3 3-hydroxyoctanoic acid (%) 2.4 3-hydroxydecanoic acid (%)3.3 3-hydroxydodecanoic acid (%) 0.7 3-hydroxydodecenoic acid (%) 1.53-hydroxy-5-phenoxyvaleric acid (%) 91.6

TABLE 78 Cell (Dry Weight) (mg/L) 910 Polymer (Dry Weight) (mg/L) 390Number Average Molecular Weight (Mn) × 10⁴ 9.1 Weight Average MolecularWeight (Mw) × 10⁴ 21.4 3-hydroxyhexanoic acid (%) 0.2 3-hydroxyoctanoicacid (%) 2.2 3-hydroxydecanoic acid (%) 4.9 3-hydroxydodecanoic acid (%)0.8 3-hydroxydodecenoic acid (%) 1.5 3-hydroxy-5-phenoxyvaleric acid (%)90.4

TABLE 79 Cell (Dry Weight) (mg/L) 1400 Polymer (Dry Weight) (mg/L) 935Number Average Molecular Weight (Mn) × 10⁴ 6.2 Weight Average MolecularWeight (Mw) × 10⁴ 14.0 3-hydroxyoctanoic acid (%) 0.6 3-hydroxydecanoicacid (%) 0.7 3-hydroxy-5-phenylvaleric acid (%) 98.7

TABLE 80 Cell (Dry Weight) (mg/L) 1350 Polymer (Dry Weight) (mg/L) 955Number Average Molecular Weight (Mn) × 10⁴ 6.1 Weight Average MolecularWeight (MW) × 10⁴ 13.8 3-hydroxyoctanoic acid (%) 1.93-hydroxy-5-phenylvaleric acid (%) 98.1

TABLE 81 Cell (Dry Weight) (mg/L) 2050 Polymer (Dry Weight) (mg/L) 1310Number Average Molecular Weight (Mn) × 10⁴ 6.3 Weight Average MolecularWeight (MW) × 10⁴ 13.9 3-hydroxybutyric acid (%) 7.5 3-hydroxyoctanoicacid (%) 0.8 3-hydroxydecanoic acid (%) 0.8 3-hydroxy-5-phenylvalericacid (%) 90.6

TABLE 82 Production of Polyhydroxyalkanoate by Using strain YN23-hydroxy-4- Polymer Yield phenoxy-n-butyric (mg/L) acid unit ratioDisodium malate 20 96.6% Sodium L-glutamate 13  8.9% D(+)-glucose 898.4% n-nonanoic acid 440 ND Polypeptone 17 43.5% *GC-MS, TIC peak arearatio, ND not detected.

TABLE 83 Production of Polyhydroxyalkanoate by Using strain YN2 Cell(Dry Weight) (mg/L) 290 Polymer (Dry weight) (mg/L)  20 Monomer UnitComposition (peak area ratio) 3-hydroxybutyric acid 0.1%3-hydroxyhexanoic acid 0.2% 3-hydroxyoctanoic acid 1.1%3-hydroxynonanoic acid 0.1% 3-hydroxydecanoic acid 0.9%3-hydroxydodecanoic acid 0.2% 3-hydroxydodecenoic acid 0.5%3-hydroxy-4-phenoxy-n-butyric acid 96.9% 

1 1 1501 DNA Pseudomonas jessenii 161 strain 1 tgaacgctgg cggcaggcctaacacatgca agtcgagcgg atgacgggag cttgctcctg 60 aattcagcgg cggacgggtgagtaatgcct aggaatctgc ctggtagtgg gggacaacgt 120 ctcgaaaggg acgctaataccgcatacgtc ctacgggaga aagcagggga ccttcgggcc 180 ttgcgctatc agatgagcctaggtcggatt agctagttgg tgaggtaatg gctcaccaag 240 gcgacgatcc gtaactggtctgagaggatg atcagtcaca ctggaactga gacacggtcc 300 agactcctac gggaggcagcagtggggaat attggacaat gggcgaaagc ctgatccagc 360 catgccgcgt gtgtgaagaaggtcttcgga ttgtaaagca ctttaagttg ggaggaaggg 420 cattaaccta atacgttagtgttttgacgt taccgacaga ataagcaccg gctaactctg 480 tgccagcagc cgcggtaatacagagggtgc aagcgttaat cggaattact gggcgtaaag 540 cgcgcgtagg tggtttgttaagttggatgt gaaagccccg ggctcaacct gggaactgca 600 ttcaaaactg acaagctagagtatggtaga gggtggtgga atttcctgtg tagcggtgaa 660 atgcgtagat ataggaaggaacaccagtgg cgaaggcgac cacctggact gatactgaca 720 ctgaggtgcg aaagcgtggggagcaaacag gattagatac cctggtagtc cacgccgtaa 780 acgatgtcaa ctagccgttgggagccttga gctcttagtg gcgcagctaa cgcattaagt 840 tgaccgcctg gggagtacggccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca 900 caagcggtgg agcatgtggtttaattcgaa gcaacgcgaa gaaccttacc aggccttgac 960 atccaatgaa ctttccagagatggatgggt gccttcggga acattgagac aggtgctgca 1020 tggctgtcgt cagctcgtgtcgtgagatgt tgggttaagt cccgtaacga gcgcaaccct 1080 tgtccttagt taccagcacgtaatggtggg cactctaagg agactgccgg tgacaaaccg 1140 gaggaaggtg gggatgacgtcaagtcatca tggcccttac ggcctgggct acacacgtgc 1200 tacaatggtc ggtacagagggttgccaagc cgcgaggtgg agctaatccc acaaaaccga 1260 tcgtagtccg gatcgcagtctgcaactcga ctgcgtgaag tcggaatcgc tagtaatcgc 1320 gaatcagaat gtcgcggtgaatacgttccc gggccttgta cacaccgccc gtcacaccat 1380 gggagtgggt tgcaccagaagtagctagtc taaccttcgg gaggacggtt accacggtgt 1440 gattcatgac tggggtgaagtcgtaccaag gtagccgtag gggaacctgc ggctggatca 1500 c 1501

What is claimed is:
 1. A process of producing a polyhydroxyalkanoate,comprising a step of culturing a microorganism capable of synthesizing apolyhydroxyalkanoate of which monomer unit is represented by Formula (1)from an alkanoate in a medium containing the alkanoate:A_(m)B_((1−m))  (1)  wherein A is represented by General Formula (2), Bis at least one selected from the group consisting of monomer unitsrepresented by General Formula (3) or (4), and m is 0.01 or larger andsmaller than 1,

 wherein n is an integer selected from 0 to 10, k is 3 or 5, and R is atleast one group selected from the group consisting of the groupsrepresented by General Formulae (5) to (7):

 in Formula (5) R1 is a group selected from the group consisting of ahydrogen atom (H), halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; andq is an integer selected from 1 to 8;  in Formula (6) R2 is a groupselected from the group consisting of a hydrogen atom (H), halogenatoms, —CN, —NO₂, —CF₃, —C₂F₅ and —C₃F₇; and r is an integer selectedfrom 1 to 8;  in Formula (7) R3 is a group selected from the groupconsisting of a hydrogen atom (H), halogen atoms, —CN, —NO₂, —CF₃, —C₂F₅and —C₃F₇; and s is an integer selected from 1 to 8; provided thatfollowing R are excluded from the choice: when selecting one type ofgroup as R in General  Formula (2): groups of Formula (5) in which R1 isH and q 2, R1 is H and q 3, and R1 is —NO₂ and q=2; the groups ofFormula (6) in which R2 is a halogen atom and r=2, R2 is —CN and r=3,and R2 is —NO₂ and r=3; and groups of Formula (7) in which R3 is H ands=1, and R3 is H and s=2; and when selecting two types of groups as R inGeneral Formula (2), groups of Formula (6) in which R2 is a halogen atomand r=2.
 2. A process of producing polyhydroxyalkanoate, comprising astep of culturing a microorganism capable of producing thepolyhydroxyalkanoate utilizing alkanoate in a medium containing thealkanoate and a saccharide.
 3. The process according to claim 2, whereinthe alkanoate is represented by Formula (22) and thepolyhydroxyalkanoate comprises a monomer unit of Formula (23):

wherein R is at least one group selected from the group consisting ofgroups of Formula (24):

 wherein R4 represents a substituted or unsubstituted phenyl group, asubstituted or unsubstituted phenoxy group, or a substituted orunsubstituted cyclohexyl group, and t is an integer of 1 to 8independently;

 wherein R′ is at least one group selected from the group consisting ofthe selected R and groups represented by Formula (24) where R₄ is thesame as the selected R but t is shorter by multiply of 2 than t of theselected R and not
 0. 4. The process according to claim 2, wherein themicroorganism is cultured in one step in a medium containing thealkanoate of Formula (22) and a saccharide.
 5. The process according toclaim 2, wherein the microorganism is cultured in at least two steps:one is in a medium containing the alkanoate of Formula (22) and asaccharide and the subsequent one is in a medium containing thealkanoate of Formula (22) and a saccharide with nitrogen sourcelimitation.
 6. The process according to claim 2, wherein themicroorganism is cultured by inoculating the microorganism preculturedin a medium containing a saccharide.
 7. The process according to claim2, wherein the saccharide is at least one selected from the groupconsisting of glucose, fructose and mannose.
 8. A process of producingpolyhydroxyalkanoate, comprising a step of culturing a microorganismcapable of producing a polyhydroxyalkanoate utilizing an alkanoate in amedium containing the alkanoate and a polypeptone.
 9. The processaccording to claim 8, wherein the alkanoate is represented by Formula(22) and the polyhydroxyalkanoate comprises a monomer unit of Formula(23):

wherein R is at least one group selected from the group consisting ofthe groups of Formula (24):

 wherein R4 represents a substituted or unsubstituted phenyl group, asubstituted or unsubstituted phenoxy group, or a substituted orunsubstituted cyclohexyl group, and t is an integer of 1 to 8independently

 wherein R′ is at least one group selected from the group consisting ofthe selected R and groups represented by Formula (24) where R₄ is thesame as the selected R but t is shorter by multiply of 2 than t of theselected R and not
 0. 10. The process according to claim 8, wherein themicroorganism is cultured in one step in a medium containing thealkanoate of Formula (22) and polypeptone.
 11. The process according toclaim 8, wherein the microorganism is cultured in at least two steps:one is in a medium containing an kanoate represented by Formula (22) andpolypeptone, and the subsequent one is in a medium containing thealkanoate with nitrogen source limitation.